O-alkyl-benzylideneguanidine derivatives and therapeutic use for the treatment of disorders associated an accumulation of misfolded proteins

ABSTRACT

The present invention relates to a compound of formula (I), or a tautomer and/or a pharmaceutically acceptable salt thereof Formula (I), and its uses to treat a disorder associated with protein misfolding stress and in particular with an accumulation of misfolded proteins.

The present invention relates to compounds that have potentialtherapeutic applications in treating disorders associated with proteinmisfolding stress and in particular with an accumulation of misfoldedproteins. In particular, the invention provides compounds that arecapable of exhibiting a protective effect against cytotoxic endoplasmicreticulum (ER) stress.

BACKGROUND TO THE INVENTION

The compound 2-(2,6-dichlorobenzylidene)hydrazinecarboximidamide, alsoreferred to as guanabenz, is an alpha agonist of the alpha-2 type thatis used as an antihypertensive drug.

Various derivatives of guanabenz have also been reported. For example,U.S. Pat. No. 3,982,020 (Sandoz, Inc.) discloses substituted benzylidenehydrazines and their use as hypoglycemic-antihyperglycemic agents,anti-obesity agents and anti-inflammatory agents. US 2004/0068017(Bausch & Lomb Inc.) discloses substituted benzylidene hydrazines thatare capable of increasing the activity of gelatinase A in ocular cells.The molecules have applications in the treatment of primary open angleglaucoma. WO 2008/061647 (Acure Pharma AB) discloses the use ofN-(2-chloro-3,4,-dimethoxybenzylideneamino)guanidine as a VEGFRinhibitor and its associated applications in the treatment or preventionof undesired blood vessel formation during tumour growth and/orinflammatory conditions. WO2005/031000 (Acadia Pharmaceuticals, Inc.)discloses substituted benzylidene hydrazines and their use in treatingacute pain and chronic neuropathic pain. Finally, EP1908464 (CNRS)discloses guanabenz and chloroguanabenz and their use in the treatmentof polyglutamine expansion associated diseases, including Huntington'sdisease.

More recently it has been reported that guanabenz has therapeuticpotential in a number of other areas. Guanabenz, was recently noted tohave anti-prion activity (Tribouillard-Tanvier et al., 2008 PLoS One 3,e1981). It has been reported that its activity in protecting againstprotein misfolding is surprisingly much broader and includes attenuatingaccumulation of mutant Huntingtin in cell-based assays (WO2008/041133)and protection against the lethal effects of expression of misfoldingprone Insulin Akita mutant in the endoplasmic reticulum (ER) of Min6 andINS-1 pancreatic beta-cells (Tsaytler et al., 2011 Science 332 pp91-94). WO2014/138298 and Way et al. (2015 Nature Communications 6:6532DOI: 10.1038/ncomms7532) disclose guanabenz ant its use in the treatmentof demyelinating disorder, such as multiple sclerosis.

Guanabenz has also been shown to promote survival of HeLa cells exposedto otherwise cytotoxic ER-stress induced by the N-glycosylationinhibitor tunicamycin, in a dose-dependent manner (Tsaytler, et al.,Science, 2011). Quantitative assessment of cell viability revealed thatguanabenz doubled the number of cells surviving ER stress with a medianeffective concentration of ˜0.4 μM. Neither the α2-adrenergic receptoragonist clonidine, nor the α2-adrenergic receptor antagonist efaroxanprotected cells from cytotoxic ER stress and efaroxan did not interferewith guanabenz's protective effect (Tsaytler, et al., Science, 2011).These observations demonstrate that guanabenz rescues cells from lethalER stress by a mechanism independent of the α2-adrenergic receptor.Guanabenz protects cells from otherwise lethal accumulation of misfoldedproteins by binding to a regulatory subunit of protein phosphatase 1,PPP1R15A (GADD34), selectively disrupting the stress-induceddephosphorylation of the a subunit of translation initiation factor 2(eIF2α). Guanabenz sets the translation rates in stressed cells to alevel manageable by available chaperones, thereby restoring proteinhomeostasis. It was reported that Guanabenz does not bind to theconstitutive PPP1R15B (CReP) and therefore does not inhibit translationin non-stressed cells (Tsaytler, et al., Science, 2011).

Failure to maintain proteostasis in the ER by mounting an adequateunfolded protein response (UPR) is recognized as a contributing factorto many pathological conditions. Thus, the molecules described here,which inhibit elF2α phosphatase to fine-tune protein synthesis, may beof therapeutic benefit to a large number of diseases caused proteinmisfolding stress and in particular with an accumulation of misfoldedproteins. Tribouillard-Tanvier et al., PLoS One 3, e1981 (2008) andEP1908464A disclose benzylidene guanidine derivatives comprisingguanidine as a terminal group. However, the applicant has found that theterminal group is liable to metabolization which affects thebiavailability of the coumpounds. Further, previous studies have alsoindicated that the (hetero)aryl group must be at least di-halogenated inorder for the compounds to exhibit useful pharmacological activity (seefor example, Tribouillard-Tanvier et al., PLoS One 3, e1981 (2008) andEP1908464A, CNRS). However, contrary to the results of previous studies,the present Applicant has surprisingly found that mono-halogenated(hetero)aryl derivatives comprising a modified terminal group may alsobe active. It is thus desirable to provide alternative, with enhancedactivity and/or bioavailability profile.

The present invention seeks to provide alternative compounds based on aguanabenz core structure that have potential therapeutic applications intreating disorders associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins.

STATEMENT OF INVENTION

A first aspect of the invention relates to a compound of formula (I), ora pharmaceutically acceptable salt thereof,

wherein:

Hal=F, Cl, Br, I

X is either —CR1= or —N═,

Y is either —CR2= or —N═,

Z is either —CR3= or —N═,

W is either —CR4= or —N═,

R1 is selected from H, Hal, alkyl and O-alkyl;

R2 is selected from H, Hal, alkyl, O-alkyl and C(O)R6;

R3 is selected from H, Hal, alkyl and O-alkyl;

R4 is H, Cl, F, I or Br;

R5 is H or alkyl, cycloalkyl, aralkyl, alkenyl, cycloalkenyl,heterocyclyl, aryl, C(O)-alkyl, and C(O)-aryl, each of which isoptionally substituted with one or more R7 groups; R6 is selected fromOH, O-alkyl, O-aryl, aralkyl, NH₂, NH-alkyl, N(alkyl)₂, NH-aryl, CF₃,alkyl and alkoxy;

each R7 is independently selected from halogen, OH, CN, COO-alkyl,aralkyl, heterocyclyl, S-alkyl, SO-alkyl, SO₂-alkyl, SO₂-aryl, COOH,CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyl and alkoxy.

And wherein if Hal is Cl and R4 is Cl, then R5 is not H.

A second aspect of the invention relates to a pharmaceutical compositioncomprising a compound of formula (II):

wherein:

Hal=F, Cl, Br, I

X is either —CR1= or —N═,

Y is either —CR2= or —N═,

Z is either —CR3= or —N═,

W is either —CR4= or —N═,

R1 is selected from H, Hal, alkyl and O-alkyl;

R2 is selected from H, Hal, alkyl, O-alkyl and C(O)R6;

R3 is selected from H, Hal, alkyl and O-alkyl;

R4 is H, Cl, F, I or Br;

R5 is H or alkyl, cycloalkyl, aralkyl, alkenyl, cycloalkenyl,heterocyclyl, aryl, C(O)-alkyl, and C(O)-aryl, each of which isoptionally substituted with one or more R7 groups;

R6 is selected from OH, O-alkyl, O-aryl, aralkyl, NH₂, NH-alkyl,N(alkyl)₂, NH-aryl, CF₃, alkyl and alkoxy;

each R7 is independently selected from halogen, OH, CN, COO-alkyl,aralkyl, heterocyclyl, Salkyl, SO-alkyl, SO₂-alkyl, SO₂-aryl, COOH,CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyl and alkoxy;

with a suitable pharmaceutically acceptable diluent, excipient orcarrier.

A third aspect of the invention relates to a compound of formula (II),or a pharmaceutically acceptable salt thereof, for use in treating adisorder associated with protein misfolding stress in particular withaccumulation of misfolded proteins, more specifically to proteopathies:

wherein:

Hal=F, Cl, Br, I

X is either —CR1= or —N═,

Y is either —CR2= or —N═,

Z is either —CR3= or —N═,

W is either —CR4= or —N═,

R1 is selected from H, Hal, alkyl and O-alkyl;

R2 is selected from H, Hal, alkyl, O-alkyl and C(O)R6;

R3 is selected from H, Hal, alkyl and O-alkyl;

R4 is H, Cl, F, I or Br;

R5 is H or alkyl, cycloalkyl, aralkyl, alkenyl, cycloalkenyl,heterocyclyl, aryl, C(O)-alkyl, and C(O)-aryl, each of which isoptionally substituted with one or more R7 groups;

R6 is selected from OH, O-alkyl, O-aryl, aralkyl, NH₂, NH-alkyl,N(alkyl)₂, NH-aryl, OF₃, alkyl and alkoxy;

each R7 is independently selected from halogen, OH, CN, COO-alkyl,aralkyl, heterocyclyl, S-alkyl, SO-alkyl, SO₂-alkyl, SO₂-aryl, COOH,CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, OF₃, alkyl and alkoxy.

And a pharmaceutically acceptable excipient.

Formula (I) is a particular embodiment of formula (II).

In a preferred embodiment, compounds of formula (I) or (II) as definedabove advantageously exhibit no activity toward the adrenergic α2Areceptor relative to prior art compounds such as Guanabenz. This loss inalpha-2 adrenergic activity renders the compounds therapeutically usefulin the treatment of the disorders associated with protein misfoldingstress and in particular with an accumulation of misfolded proteins. Theabsence of alpha-2 adrenergic activity means that compounds of formula(I) or (II) can be administered at a dosage suitable to treat theaforementioned diseases, without any significant effect on bloodpressure.

DETAILED DESCRIPTION

As used herein, the term “alkyl” includes both saturated straight chainand branched alkyl groups. Preferably, the alkyl group is a C₁₋₂₀ alkylgroup, more preferably a C₁₋₁₅, more preferably still a C₁₋₁₂ alkylgroup, more preferably still, a C₁₋₆ alkyl group, more preferably a C₁₋₃alkyl group. Particularly preferred alkyl groups include, for example,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyland hexyl.

As used herein, the term “cycloalkyl” refers to a cyclic alkyl group.Preferably, the cycloalkyl group is a C₃₋₁₂ cycloalkyl group.

As used herein, the term “alkenyl” refers to a group containing one ormore carbon-carbon double bonds, which may be branched or unbranched.Preferably the alkenyl group is a C₂₋₂₀ alkenyl group, more preferably aC₂₋₁₆ alkenyl group, more preferably still a C₂₋₁₂ alkenyl group, orpreferably a C₂₋₆ alkenyl group, more preferably a C₂₋₃ alkenyl group.The term “cyclic alkenyl” is to be construed accordingly.

As used herein, the term “aryl” refers to a C₆₋₁₂ aromatic group.Typical examples include phenyl and naphthyl etc.

As used herein, the term “heterocycle” (also referred to herein as“heterocyclyl” and “heterocyclic”) refers to a 4 to 12, preferably 4 to6 membered saturated, unsaturated or partially unsaturated cyclic groupcontaining one or more heteroatoms selected from N, O and S, and whichoptionally further contains one or more CO groups. The term“heterocycle” encompasses both heteroaryl groups and heterocycloalkylgroups as defined below.

As used herein, the term “heteroaryl” refers to a 4 to 12 memberedaromatic which comprises one or more heteroatoms. Preferably, theheteroaryl group is a 4 to 6 membered aromatic group comprising one ormore heteroatoms selected from N, O and S. Suitable heteroaryl groupsinclude pyrrole, pyrazole, pyrimidine, pyrazine, pyridine, quinoline,thiophene, 1,2,3-triazole, 1,2,4-triazole, thiazole, oxazole,iso-thiazole, iso-oxazole, imidazole, furan and the like.

As used herein, the term “heterocycloalkyl” refers to a 3 to 12membered, preferably 4 to 6 membered cyclic aliphatic group whichcontains one or more heteroatoms selected from N, O and S. N-containing5 to 6 membered heterocycloalkyl are preferred. Preferredheterocycloalkyl groups include piperidinyl, pyrrolidinyl, piperazinyl,thiomorpholinyl and morpholinyl. More preferably, the heterocycloalkylgroup is selected from N-piperidinyl, N-pyrrolidinyl, N-piperazinyl,N-thiomorpholinyl and N-morpholinyl.

As used herein, the term “aralkyl” includes, but is not limited to, agroup having both aryl and alkyl functionalities. By way of example, theterm includes groups in which one of the hydrogen atoms of the alkylgroup is replaced by an aryl group, e.g. a phenyl group. Typical aralkylgroups include benzyl, phenethyl and the like.

The followings are particular embodiments of formula (I) or (II):

In one preferred embodiment, Hal is Cl.

In one preferred embodiment, X is —CR1=.

In one preferred embodiment Y is —CR2=.

In another preferred embodiment, Y is N.

In one preferred embodiment Z═—CR3=.

In one preferred embodiment W═—CR4=.

In one preferred embodiment, R1 is H or F, more preferably H.

In one preferred embodiment, R2 is H or F, more preferably H.

In one preferred embodiment, R3 is H or F more preferably H.

In one preferred embodiment, R4 is H, Cl or F preferably H or F morepreferably H.

In one preferred embodiment, R3 and R4 are both H.

In one embodiment, R5 is H, alkenyl or alkyl, each of alkenyl or alkylbeing optionally substituted with one or more R7 groups.

In one embodiment, R7 groups are chosen from halogen, OH, heterocyclyl,SO-alkyl, SO₂-alkyl, Oalkyl.

In one especially preferred embodiment, the compound of formula (I) or(II) is selected from the following:

and pharmaceutically acceptable salts thereof.

In a preferred embodiment, the compound of formula (I) or (II) isselected from Compounds 4, 6, 10, 11, 12, 14, 17, 18 as set out above,more preferably selected from Compounds 4, 11, 17, 18 as set out above.

Compounds

One aspect of the invention relates to compounds of formulae (I), orpharmaceutically acceptable salts thereof, as defined above. Preferredaspects of the invention apply mutatis mutandis. Particularly preferredcompounds for this aspect of the invention include Compounds 1, 2, 5 and10 as described herein.

Process of Preparation

A further aspect of the invention relates to a process for preparing acompound of formula (I) or (II) or pharmaceutically acceptable saltsthereof as above described, comprising the step of reacting a compoundof formula (A) or a tautomer form thereof:

wherein R5 is as defined above

with a compound of formula (B):

wherein X, Y, Z, W and Hal are as defined above,

optionally followed by a step of modifying the R5 group of the compoundresulting from the reaction between the compounds of formulae (A) and(B) as above described, into another R5 group.

Preferably, the process may also comprise a further step of purificationof the compound (I) or (II), obtained above.

The coupling reaction between compounds (A) and (B) may be conducted inan organic solvent, such as an alcohol, eg ethanol. It may be carriedout at a temperature comprised between room temperature and the boilingtemperature of the reaction mixture.

The modification reaction of R5 groups may be conducted by applicationor adaptation of known methods. For example, in the compound obtainedfollowing the coupling of (A) and (B), R5 may be an alkyl groupsubstituted by R7 groups: it may thus be desired to substitute R7groups. Such substitution reactions are generally known. As arepresentative examples it may be desired to replace R7=OH withR7=halogen in a compound of formula (I) or (II). Such reaction may beconducted in the presence of an halogenating agent, such as achlorinating agent, eg SOCl₂. Typically such a reaction may beconducting in an organic solvent such as dichloromethane. Anotherrepresentative example is the substitution of R7=halogen withR7=N-containing heterocycle such as pyrrolidine. Such reaction may beconducted in the presence of a base, such as TEA. Typically such areaction may be conducting in an organic solvent such as THF.

According to an embodiment, the process may further comprise the step ofpreparing the compound of formula (A) as above defined by reacting acompound of formula (C):

or one of its salts

wherein R5 is as defined above

with the S-methylisothiosemicarbazide hydroiodide compound (D):

where Lg is a leaving group such as —S-Alkyl, e.g. —S-Methyl.

or one of its salts.

Typically, the reaction between the compounds of formulae (C) and (D)may be carried out in a basic aqueous solution, for example in anaqueous solution comprising sodium hydroxide.

The coupling reaction between compounds of formulae (C) and (D) may befollowed a further step of purification.

In an embodiment, the process may optionally comprise a further step ofpreparing the compound of formula (C) by reacting a compound of formula(E):

with a hydrazine derivative compound, for example hydrazine hydrate ormethyl hydrazine.

The process of the invention may optionally comprise the step ofpreparing the compound of formula (E) from a compound of formula (E′):

Where (R5′) represents a precursor group of R5.

This reaction may be desired when (E) is not commercially available andit is not practicable to prepare (E) from (F) and (G) as disclosedbelow.

It may thus be desirable to use a precursor (E′) which is to betransformed into (E).

A precursor is a group or a compound that may be modified into thedesired compound by a substitution, elimination or otherwise derivationchemical reaction.

As an illustrative embodiment, the modification reaction of a R5′ intothe desired R5 group may be conducted by application or adaptation ofknown methods. For example, in (E), R5 may be an alkyl group substitutedby R7 groups: it may thus be desired to modify R7′ groups in (E′) intothe desired R7′ in (E). Such modification reactions are generally known.As a representative example, it may be desired to replace the precursorR5′ comprising the group R7′=S(Alkyl) with R7=SO₂(Alkyl). Such reactionmay be conducted in the presence of MCPBA. Typically such a reaction maybe conducting in an organic solvent such as dichloromethane.

The process of the invention may comprise the step of preparing (E) or(E′) as appropriate, by reacting a compound (F)

Lg′-R5″   (F)

Where R5″ represents either R5 or R5′ as defined above, and Lg′represents a leaving group such as a halogen atom or a hydroxyl (OH)group, with N-hydroxyphtalimide (G):

Generally, the coupling of (F) and (G) may be conducted according to aGabriel synthesis conditions.

According to an illustrative embodiment, this reaction may be carriedout in the presence of a base such as organic or mineral base, typicallyTEA or K₂CO₃, or NaOAc, in particular where Lg contains Halogen(s).

According to another illustrative embodiment, the first step may becarried out in the presence of diisopropyl azodicarboxylate and PPh3, inparticular where Lg=OH.

Compounds (F), (G), (B) are generally commercially available.

The compounds of formula (D):

where Lg is a —S-Alkyl, e.g. —S-Methyl is also part of the invention.

In addition to the process disclosed above, the compounds and process ofthe present invention may be prepared in a number of ways well known tothose skilled in the art. The compounds can be synthesized, for example,by application or adaptation of the methods described below, orvariations thereon as appreciated by the skilled artisan.

The appropriate modifications and substitutions will be readily apparentand well known or readily obtainable from the scientific literature tothose skilled in the art.

In particular, such methods can be found in R. C. Larock, ComprehensiveOrganic Transformations, VCH publishers, 1989

It will be appreciated that the compounds of the present invention maycontain one or more asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. Thus, all chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated. It is well known in the art how toprepare and isolate such optically active forms. For example, mixturesof stereoisomers may be separated by standard techniques including, butnot limited to, resolution of racemic forms, normal, reverse-phase, andchiral chromatography, preferential salt formation, recrystallization,and the like, or by chiral synthesis either from chiral startingmaterials or by deliberate synthesis of target chiral centers.

Compounds of the present invention may be prepared by a variety ofsynthetic routes. The reagents and starting materials are commerciallyavailable, or readily synthesized by well-known techniques by one ofordinary skill in the arts. All substituents, unless otherwiseindicated, are as previously defined.

In the reactions described herein, it may be necessary to protectreactive functional groups, for example hydroxy, amino, imino, thio orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Conventional protectinggroups may be used in accordance with standard practice, for examplessee T. W. Greene and P. G. M. Wuts in Protective Groups in OrganicSynthesis, John Wiley and Sons, 1991; J. F. W. McOmie in ProtectiveGroups in Organic Chemistry, Plenum Press, 1973.

Some reactions may be carried out in the presence of a base. There is noparticular restriction on the nature of the base to be used in thisreaction, and any base conventionally used in reactions of this type mayequally be used here, provided that it has no adverse effect on otherparts of the molecule. Examples of suitable bases include: sodiumhydroxide, potassium carbonate, triethylamine, alkali metal hydrides,such as sodium hydride and potassium hydride; alkyllithium compounds,such as methyllithium and butyllithium; and alkali metal alkoxides, suchas sodium methoxide and sodium ethoxide.

Usually, reactions are carried out in a suitable solvent. A variety ofsolvents may be used, provided that it has no adverse effect on thereaction or on the reagents involved. Examples of suitable solventsinclude: hydrocarbons, which may be aromatic, aliphatic orcycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene,toluene and xylene; amides, such as dimethyl-formamide; alcohols such asethanol and methanol and ethers, such as diethyl ether andtetrahydrofuran.

The reactions can take place over a wide range of temperatures. Ingeneral, we find it convenient to carry out the reaction at atemperature of from 0° C. to 150° C. (more preferably from about roomtemperature to 100° C.). The time required for the reaction may alsovary widely, depending on many factors, notably the reaction temperatureand the nature of the reagents. However, provided that the reaction iseffected under the preferred conditions outlined above, a period of from3 hours to 20 hours will usually suffice.

The compound thus prepared may be recovered from the reaction mixture byconventional means. For example, the compounds may be recovered bydistilling off the solvent from the reaction mixture or, if necessaryafter distilling off the solvent from the reaction mixture, pouring theresidue into water followed by extraction with a water-immiscibleorganic solvent and distilling off the solvent from the extract.Additionally, the product can, if desired, be further purified byvarious well-known techniques, such as recrystallization,reprecipitation or the various chromatography techniques, notably columnchromatography or preparative thin layer chromatography.

The process of the invention may also include the additional step ofisolating the obtained product of formula (I).

The starting products and/or reagents may be commercially available, ormay be readily prepared by the skilled person by applying or adaptingthe procedures disclosed in the experimental part below.

Therapeutic Applications

The compounds of formula (I) or (II) have potential therapeuticapplications in treating disorders associated with accumulation ofmisfolded and/or unfolded proteins. In particular, compounds of formula(I) or (II) may have a protective effect against cytotoxic endoplasmicreticulum (ER) stress and age related disorders.

Another aspect of the invention relates to the use of a compound offormula (I) or (II) as defined above in the preparation of a medicamentfor treating a disorder associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins.

In one preferred embodiment of the invention, the compound of formula(I) or (II) is for use in treating diseases where accumulation ofmisfolded and/or unfolded proteins is involved in the mode of action(Brown et al, 2012, Frontiers in Physiology, 3, Article 263).

Another aspect of the invention relates to the use of a compound offormula (I) or (II) as defined above in the preparation of a medicamentfor treating proteopathies. The proteopathies refer to a class ofdiseases in which certain proteins become structurally abnormal, andthereby disrupt the function of cells, tissues and organs of the body.Often the proteins fail to fold into their normal conformation, and inthis misfolded and/or unfolded state, the proteins can become toxic insome way (a gain of toxic function) or they can lose their normalfunction or they can have a reduce biological activity. Theproteopathies, also known as proteinopathies, protein conformationaldisorders, or protein misfolding diseases, include many diseases suchdiseases as Alzheimer's disease, Parkinson's disease, prion disease,type 2 diabetes, amyloidosis, and a wide range of other disorders (seenon limiting examples below).

As used herein the terms “proteinopathies, proteopathies, proteinconformational disorders, protein misfolding diseases, diseasesassociated with protein misfolding stress, diseases associated with anaccumulation of misfolded protein, diseases associated with a cytotoxicER stress, UPR related diseases associated with have the same meaningand refer to diseases wherein certain protein become structurallyabnormal and thereby disrupt the cellular homeostasis.

As used herein the terms “misfolded protein” and “unfolded protein” hasthe same meaning and refer to protein that fail to fold into theirnormal conformation.

As used herein the phrase “preparation of a medicament” includes the useof one or more of the above described compounds directly as themedicament in addition to its use in a screening programme for furtheractive agents or in any stage of the manufacture of such a medicament.

Yet another aspect of the invention relates to a method of treatingproteinopathy and/or a disorder associated with protein misfoldingstress and in particular with an accumulation of misfolded proteins in asubject in need thereof, said method comprising administering atherapeutically effective amount of a compound of formula (I) or (II) asdefined above to said subject.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

Herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease ordisorder, substantially ameliorating clinical symptoms of a disease ordisorder or substantially preventing the appearance of clinical symptomsof a disease or disorder.

As used herein, the term <<disease>>, <<disorder>>, <<conditions>> hasthe same meaning. The disease is associated with an ER stress responseactivity and/or is associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins.

The term “therapeutically effective amount” refers to that amount of thecompound being administered which will relieve to some extent one ormore of the symptoms of the disease or disorder being treated.

In another embodiment, the invention relates to a compound of formula(I) or (II) as defined above for use in treating UPR disorders. The term“unfolded protein response” or UPR refers to a component of the cellulardefence system against misfolded proteins that adapts folding in theendoplasmic reticulum (ER) to changing conditions. The UPR is activatedin response to an accumulation of unfolded or misfolded proteins in thelumen of the endoplasmic reticulum. In this scenario, the UPR has twoprimary aims: (i) to restore normal function of the cell by haltingprotein translation, and (ii) to activate the signaling pathways thatlead to the increased production of molecular chaperones involved inprotein folding. If these objectives are not achieved within a certaintime frame, or the disruption is prolonged, the UPR aims towardsapoptosis. Upstream components of the UPR are the ER-residenttrans-membrane proteins IRE1, ATF6, and PERK, which sense foldingdefects to reprogram transcription and translation in a concerted mannerand restore proteostasis. Activated IRE1 and ATF6 increase thetranscription of genes involved in ER folding, such as those encodingthe chaperones BiP and GRP94. Activated PERK attenuates global proteinsynthesis by phosphorylating the subunit of translation initiationfactor 2 (eIF2α) on Ser51 while promoting translation of thetranscription factor ATF4. The latter controls expression of CHOP,another transcription factor, which in turn promotes expression ofPPP1R15A/GADD34. PPP1R15A, an effector of a negative feedback loop thatterminates UPR signaling, recruits a catalytic subunit of proteinphosphatase 1 (PP1c) to dephosphorylate elF2α, allowing proteinsynthesis to resume. UPR failure contributes to many pathologicalconditions that might be corrected by adequate boost of this adaptiveresponse. Selective inhibitors of the stressed-induced elF2α phosphatasePPP1R15A-PP1 delays elF2α dephosphorylation and consequently proteinsynthesis selectively in stressed cells, without affecting proteinsynthesis in unstressed cells which constitutively expresses elF2αphosphatase PPP1R15B-PP1. This prolongs the beneficial effects of theUPR. A transient reduction of protein synthesis is beneficial tostressed cells because decreasing the flux of proteins synthetizedincreases the availability of chaperones and thus protects frommisfolding stress (Tsaytler et al., 2011 Science, 332, 91-94).Non-selective inhibitors of the 2 elF2α phosphatases PPP1R15A-PP1 andPPP1R15B-PP1 might have undesirable effects, as persistent translationinhibition is deleterious. Indeed, genetic ablation of both PPP1R15A andPPP1R15B results in early embryonic lethality in mice indicating thatinhibition of the two elF2α phosphatases PPP1R15A-PP1 and PPP1R15B-PP1is deleterious in an organismal context. In contrast, genetic ablationof PPP1R15A has no harmful consequence in mice (Harding et al., 2009,Proc. Natl. Acad. Sci. USA, 106, 1832-1837). Furthermore, specificinhibitors of PPP1R15A are predicted to be inert in unstressed cells, asthe PPP1R15A is not expressed in absence of stress. Thus, selectivePPP1R15A inhibitors are predicted to be safe. Non-selective inhibitorsof the two elF2α phosphatases may also be useful to treat proteinmisfolding diseases, when used at doses that result in only a partialinhibition of the phosphatases.

Cytoprotection against ER stress can be measured by a suitable assay.For example, cytoprotection can be measured in HeLa cells in which ERstress is elicited by the addition of media containing tunicamycin, amixture of homologous nucleoside antibiotics that inhibits theUDP-HexNAc: polyprenol-P HexNAc-1-P family of enzymes and is used toinduce unfolded protein response. Cell viability can be detected in thepresence and absence of inhibitor compounds after a set period of time,by measuring the reduction of WST-8 into formazan using a standard cellviability kit (such as Cell Viability Counting Kit-8 from Dojindo).Cytoprotection from ER stress is measured in terms of the percentageincrease in viable cells (relative to control) after ER stress. Furtherdetails of a suitable assay are set forth in the accompanying Examplessection.

In one preferred embodiment, the compound of formula (I) or (II) iscapable of prolonging the protective effect of the UPR relative to thecontrol (i.e. in the absence of inhibitor compound) by at least 10%, byat least 20%, more preferably, at least 30%, even more preferably, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, morepreferably still, at least 90%.

The compounds of formula (I) or (II) are inhibitors of PPP1R15A-PP1interaction which induce a protective effect. Preferably, the compoundexhibits a protective effect with EC₅₀ of less than about 5 μM, evenmore preferably, less than about 2 μM, more preferably still, less thanabout 1 μM. The compound should preferably be devoid of alpha2adrenergic activity. Thus, in one preferred embodiment the compound doesnot exhibit any activity in a functional alpha-2-adrenergic assay.

Certain compounds of formula (I) or (II) selectively inhibitPPP1R15A-PP1, and thus prolong the protective effect of the UPR, therebyrescuing cells from protein misfolding stress. Inhibitors ofPPP1R15A-PP1 described in the present invention therefore havetherapeutic applications in the treatment of a variety of diseasesassociated with protein misfolding stress and in particular with anaccumulation of misfolded proteins, more specifically in the treatmentof proteinopathies.

In one embodiment, the compound of formula (I) or (II) is capable ofinhibiting PPP1R15A and PPP1R15B. In one preferred embodiment, thecompound of formula (I) or (II) is capable of selectively inhibitingPPP1R15A over PPP1R15B.

In one embodiment, the invention relates to a compound of formula (I) or(II) as defined above for use in treating a disorder associated with theelF2α phosphorylation pathway where accumulation of misfolded proteinsis involved in the mode of action. Preferably, the disorder is aPPP1R15A-related disease or disorder.

In another embodiment, the invention relates to a compound of formula(I) or (II) as defined above for use in treating a disorder caused by,associated with or accompanied by elF2α phosphorylation and/or PPP1R15Aactivity where accumulation of misfolded proteins is involved in themode of action.

In another embodiment, the invention relates to a compound of formula(I) or (II) as defined above for use in treating UPR disorder such as,but not limited to aging (Naidoo et al., 2008, J Neurosci, 28, 6539-48).

As used herein, “PPP1R15A related disease or disorder” refers to adisease or disorder characterized by abnormal PPP1R15A activity whereaccumulation of misfolded proteins is involved in the mode of action.Abnormal activity refers to: (i) PPP1R15A expression in cells whichnormally do not express PPP1R15A; (ii) increased PPP1R15A expression;or, (iii) increased PPP1R15A activity.

In another embodiment, the invention relates to a method of treating amammal having a disease state alleviated by the inhibition of PP1R15A,where accumulation of misfolded proteins is involved in the mode ofaction, wherein the method comprises administering to a mammal atherapeutically effective amount of a compound of formula (I) or (II) asdefined above.

In another embodiment, the invention relates to a PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating disorders associated with protein misfolding stress andin particular with an accumulation of misfolded proteins and/or UPRdisorders, wherein said compound has no or reduced adrenergic alpha 2agonist activity in comparison with Guanabenz.

In another embodiment, the invention relates to a PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating disorders associated with protein misfolding stress andin particular with an accumulation of misfolded proteins and/or UPRdisorders, wherein said compound does not inhibit protein translation innon-stressed cells expressing PPP1R15B.

In another embodiment, the invention relates to a method of treating adisorder characterized by ER stress response activity with anaccumulation of misfolded proteins, the method comprising administeringto a patient a therapeutically effective amount of at least one compoundof formula (I) or (II) wherein said compound modulates ER stressresponse.

In another embodiment, the invention relates to PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating disorders associated with protein misfolding stress andin particular with an accumulation of misfolded proteins and/or UPRdisorders, wherein said compound has a selectivity towards PPP1R15A-PP1holophosphatase, having but no or reduced activity towards PPP1R15B-PP1holophosphatase, and wherein the ratio (activity towards PPP1R15A-PP1holophosphatase/activity towards PPP1R15B-PP1) for said compound is atleast equal or superior to the ratio (activity towards PPP1R15A-PP1holophosphatase/activity towards PPP1R15B-PP1) for Guanabenz.

In another embodiment, the invention relates to a PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating disorders associated with protein misfolding stress andin particular with an accumulation of misfolded proteins and/or UPRdisorders, wherein:

-   -   said compound has an activity towards PPP1R15A-PP1        holophosphatase but no or reduced activity towards PPP1R15B-PP1        holophosphatase, and;    -   wherein the ratio (activity towards PPP1R15A-PP1        holophosphatase/activity towards PPP1R15B-PP1) for said compound        is at least equal or superior to the ratio (activity towards        PPP1R15A-PP1 holophosphatase/activity towards PPP1R15B-PP1) for        Guanabenz; and    -   wherein said compound has no or reduced adrenergic alpha 2        agonist activity in comparison with Guanabenz.

In another embodiment, the invention relates to a PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating a disease or a condition characterized with at least oneof (1) ER stress, (2) a cellular accumulation of unfolded or misfoldedprotein and (3) an UPR.

In another embodiment, the invention relates a PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating a disease in a subject characterized by or associatedwith at least one of (1) endoplasmic reticulum (ER) stress, (2) acellular accumulation of unfolded or misfolded proteins, and (3) anunfolded protein response.

The disease is associated with an ER stress response activity and/or isassociated with protein misfolding stress and in particular with anaccumulation of misfolded and/or unfolded proteins; more specificallythe disease is a proteinopathy. Non limiting examples of diseaseaccording to the invention include, but are not limited to:

-   -   Neurodegenerative diseases such as tauopathies (such as        Alzheimer's disease among others), synucleinopathies (such as        Parkinson disease among others), Huntington disease and related        polyglutamine diseases, polyalanine diseases (such as        oculo-pharyngeal muscular dystrophy), prion diseases (also named        transmissible spongiform encephalopathies), demyelination        disorders such as Charcot-Marie Tooth diseases (also named        hereditary motor and sensory neuropathy), leukodystrophies,        amyotrophic lateral sclerosis (also referred to as motor neurone        disease and as Lou Gehrig's disease) and multiple sclerosis.

Examples of tauopathies include, but are not limited to Alzheimer'sdisease, progressive supranuclear palsy, corticobasal degeneration,frontotemporal lobar degeneration (Pick's disease). FTD is aneurodegenerative disease characterized by progressive neuronal losspredominantly involving the frontal and/or temporal lobes; second onlyto Alzheimer's disease (AD) in prevalence, FTD accounts for 20% of youngonset dementia cases. The involvement of UPR in tauopathies is welldocumented (see Stoveken 2013, The Journal of Neuroscience33(36):14285-14287). Without to be bound by a theory, it is anticipatedthat compounds of the invention which are PPP1R15A inhibitors willameliorate disease manifestations of tauopathies. In one preferredembodiment, the compound of formula (I) or (II) is for use in treatingAlzheimer's disease. According to a preferred embodiment, the inventionrelates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof for the use in treating a diseaseselected among frontotemporal dementia (FTD), supranuclear palsy andcorticobasal degeneration, preferably FTD.

Examples of synucleinopathies include, but are not limited toParkinson's disease, dementia with Lewy bodies, pure autonomic failure,and multiple system atrophy. Recently, Colla et al. (J. of Neuroscience2012 Vol. 32 No 10 pp 3306-3320) demonstrated that Salubrinal a smallmolecule that increases the phosphorylation of elF2 alpha by inhibitingthe PPP1R15A mediated dephosphorylation of elF2 alpha (Boyce et al. 2005Science Vol. 307 pp 935-939), significantly attenuates diseasemanifestations in two animal models of alpha-synucleinopathy. Thecompounds of the invention which are PPP1R15A inhibitors will amelioratedisease manifestations of alpha-syncleinopathies such as Parkinson'sdisease. In one preferred embodiment, the compound of formula (I) or(II) is for use in treating alpha-syncleinopathies such as Parkinson'sdisease.

Examples of polyglutamine diseases include but are not limited toSpinobulbar muscular atrophy (or Kennedy disease), Huntington disease,Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type 1,Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3 (orMachado-Joseph disease), Spinocerebellar ataxia type 6, Spinocerebellarataxia type 7 and Spinocerebellar ataxia type 17. Guanabenz is able toattenuate the accumulation of mutant Huntingtin in cell-based assays(WO2008/041133). This finding is unexpected since mutant huntingtin iseither cytosolic or nuclear. However, there is evidence that mutanthuntingtin metabolism has previously been connected to the ER stressresponse (Nishitoh et al., 2002, Genes Dev, 16, 1345-55; Rousseau etal., 2004, Proc Natl Acad Sci USA, 101, 9648-53; Duennwald andLindquist, 2008, Genes Dev, 22, 3308-19). The findings that guanabenzprotects cells from cytotoxic ER stress and reduces mutant huntingtinaccumulation further supports the idea that there may be aspects of theER stress response that impact on mutant huntingtin accumulation.However, Guanabenz is not useful for the treatment of human proteinmisfolding diseases due to its hypotensive activity. In contrast, theGuanabenz derivative PPP1R15A inhibitors devoid of alpha2 adrenergicactivity of the invention could be useful to treat polyglutaminediseases and more specifically Huntington disease. In one preferredembodiment, the compound of formula (I) or (II) is for use in treatingHuntington's disease.

Examples of polyalanine diseases include oculo-pharyngeal musculardystrophy which is caused by poly-alanine tract in poly(A) bindingprotein nuclear 1 (PABPN1). Barbezier et al. (2011, EMBO Vol. 3 pp35-49) demonstrated that Guanabenz reduces aggregation inoculopharyngeal muscular atrophy. According to a preferred embodiment,the invention relates to a PPP1R15A inhibitor of formula (I) or (II) ora pharmaceutical acceptable salt thereof for the use in treatingpolyalanine diseases, more specifically oculopharyngeal muscularatrophy.

Examples of prion diseases of humans include but are not limited toclassic Creutzfeldt-Jakob disease, new variant Creutzfeldt-Jakob disease(nvCJD, a human disorder related to Bovine spongiform encephalopathy),Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia andkuru. Guanabenz reduces the symptoms of prion infected mice (D.Tribouillard-Tanvier et al., 2008 PLoS One 3, e1981). However, Guanabenzis not useful for the treatment of human protein misfolding diseases dueto its hypotensive activity. In contrast, the Guanabenz derivativePPP1R15A inhibitors devoid of alpha2 adrenergic activity of theinvention could be useful to treat prion diseases. According to apreferred embodiment, the invention relates to a PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating a disease selected in the group of Creutzfeldt-Jakobdisease, new variant Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia andkuru.

Demyelination disorders are characterized by a loss of oligodendrocytesin the central nervous system or Schwann cells in the peripheral nervoussystem. The phenomenon associated with a demyelination disorder ischaracterized by a decrease in myelinated axons in the central nervoussystem or peripheral nervous system. Non-limiting exemples of misfoldedproteins of a myelinating cell (including oligodendrocyte and Schwanncell) is selected from the group consisting of CC1, myelin basic protein(MBP), ceramide galactosyltransferase (CGT), myelin associatedglycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG),oligodendrocyte-myelin glycoprotein (OMG), cyclic nucleotidephosphodiesterase (CNP), myelin protein zero (MPZ), peripheral myelinprotein 22 (PMP22), Connexin 32 (Cx32), protein 2 (P2),galactocerebroside (GalC), sulfatide and proteolipid protein (PLP). MPZ,PMP22, Cx32 and P2 are preferred misfolded proteins for Schwann cells.PLP, MBP, MAG are preferred misfolded proteins for oligodendrocytes.

In certain embodiments, the demyelination disorder is selected from thegroup consisting of Charcot-Marie Tooth (CMT) diseases. CMT refers to agroup of hereditary neuropathy disorders characterized by a chronicmotor and sensory polyneuropathy. Different types of CMT were identifiedsuch as CMT1, CMT2, CMT4, CMTX and Dejerine-Sottas disease. CMT subtypesmay be further subdivided primarily on molecular genetic findings. Forexamples CMT1 is subdivided in CMT1A, 1B, 10, 1D, 1E, 1F/2E, 1X. Over a100 mutations in the gene encoding myelin protein zero (P0), asingle-pass transmembrane protein, which is the major protein producedby myelinating Schwann cells causes Charcot-Marie-Tooth neuropathy(D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9). The mutations aredominantly inherited and cause the disease through a gain of toxicfunction (D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9). Deletionof serine 63 from PO (P0S63del) causes Charcot-Marie-Tooth 1B neuropathyin humans and a similar demyelinating neuropathy in transgenic mice. Themutant protein accumulates in the ER and induces the UPR (D'Antonio etal., 2009). Genetic ablation of CHOP, a pro-apoptotic gene in the UPRrestores motor function in Charcot-Marie-Tooth mice (Pennuto et al.,2008, Neuron, 57, 393-405). The finding that PPP1R15A inhibition incells nearly abolishes CHOP expression in ER-stressed cells indicatesthat genetic or pharmacological inhibition of PPP1R15A should reducemotor dysfunction in Charcot-Marie-Tooth mice. Recently, D'Antonio etal. (2013 J. Exp. Med Vol. pp 1-18) demonstrated that P0S63del micetreated with salubrinal, regained almost normal motor capacity inrotarod analysis and was accompanied by a rescue of morphological andelectro-physiological abnormalities. Accumulation of the of CMT-relatedmutant in the ER proteins is not unique to P0S63del; at least five otherPO mutants have been identified that are retained in the ER and elicitan UPR (Pennuto et al., 2008; Saporta et al., 2012 Brain Vol. 135 pp2032-2047). In addition, protein misfolding and accumulation ofmisfolded protein in the ER have been implicated in the pathogenesis ofother CMT neuropathies as a result of mutations in PMP22 and Cx32 (Colbyet al., 2000 Neurobiol. Disease Vol. 7 pp 561-573; Kleopa et al., 2002J. Neurosci. Res. Vol. 68 pp 522-534; Yum et al., 2002 Neurobiol. Dis.Vol. 11 pp 43-52). However, Salubrinal is toxic and cannot be used totreat human patients D'Antonio et al. (2013). In contrast, the PPP1R15Ainhibitors of formula (I) or (II) are predicted to be safe and could beuseful for the treatment of CMTs, preferably CMT-1, and more preferablyCMT-1A, CMT-1B, CMT-1E, CMT-1X. In one preferred embodiment, thecompound of formula (I) or (II) is for use in treatingCharcot-Marie-Tooth diseases, preferably CMT-1, more preferably CMT-1A,CMT-1B, CMT-1E and CMT-1X. According to a preferred embodiment, theinvention relates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof for the use in treating CMT, morepreferably CMT-1 and Dejerine-Sottas disease. According to a preferredembodiment, the invention relates to a PPP1R15A inhibitor of formula (I)or (II) or a pharmaceutical acceptable salt thereof for the use intreating CMT associated with an accumulation of misfolded protein in theER. According to a preferred embodiment, the invention relates to aPPP1R15A inhibitor of formula (I) or (II) or a pharmaceutical acceptablesalt thereof for the use in treating CMT-1A. According to a preferredembodiment, the invention relates to a PPP1R15A inhibitor of formula (I)or a pharmaceutical acceptable salt thereof for the use in treatingCMT-1B. According to a preferred embodiment, the invention relates to aPPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable saltthereof for the use in treating CMT-1E. According to a preferredembodiment, the invention relates to a PPP1R15A inhibitor of formula (I)or a pharmaceutical acceptable salt thereof for the use in treatingCMT-1X. In another embodiment, the compound of formula (I) or (II) isfor use in treating CMT, more preferably for use in treating CMT-1, inassociation with at least one compound selected in the group ofD-Sorbitol, baclofen, pilocarpine, naltrexone, methimazole,mifepristone, ketoprofene and salts thereof. The compounds are combinedfor a grouped or separate administration, simultaneously orsequentially.

The invention relates to composition comprising a PPP1R15A inhibitorselected in the group of compound of formula (I) or (II), guanabenz andsalubrinal or a pharmaceutical acceptable salt thereof, and at least onemarketed compound and salts thereof, for use in the treatment ofneurodegenerative diseases, preferably CMT, more preferably CMT-1. Thedosage of compounds in the composition shall lie within the range ofdoses not above the usually prescribed doses for long term maintenancetreatment or proven to be safe on phase 3 clinical trial; the mostpreferred dosage of compounds in the combination shall corresponds toamounts for 1% up to 10% of those usually prescribes for long termmaintenance treatment.

Thus, the invention relates to composition comprising a PPP1R15Ainhibitor selected in the group of compound of formula (I) or (II),guanabenz and salubrinal or a pharmaceutical acceptable salt thereof,and a compound increasing the expression of PMP22 protein, selected inthe group of D-Sorbitol, baclofen, pilocarpine, naltrexone, methimazole,mifepristone, ketoprofene and salts thereof, for use in the treatment ofCMT, preferably CMT-1, more preferably CMT-1A more preferably CMT-1A,CMT-1B, CMT-1E and CMT-1X.

In other embodiments, the demyelination disorder is selected from thegroup consisting of leukodystrophies. Examples of leukodystrophiesinclude but are not limited to adrenoleukodystrophy (ALD), Alexanderdisease, Canavan disease, Krabbe disease, Metachromatic Leukodystrophy(MLD), Pelizaeus-Merzbacher disease (PMD), childhood ataxia with centralnervous system hypomyelination (also known as vanishing white matterdisease), CAMFAK syndrome, Refsum Disease, Cockayne Syndrome, Ver derKnapp Syndrome, Zellweger Syndrome, Guillain-Barre Syndrome (GBS),chronic inflammatory demyelinating polyneuropathy (CIDP), multifocualmotor neuropathy (MMN) and progressive supernuclear palsy, progressiveMultifocal Leuko-encephalopathy (PML), Encephalomyelitis, CentralPontine Myelolysis (CPM), Anti-MAG Disease, among others. Gow et al.(Neuron, 2002 Vol. 36, 585-596) demonstrated that the unfolded proteinresponse is activated in PMD, and show that this pathway is duplicationof, the PLP1 gene. According to a preferred embodiment, the inventionrelates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof for the use in treatingleukodystrophies, and preferably Pelizaeus-Merzbacher disease (PMD).

Amyotrophic lateral sclerosis (ALS) is referred to as motor neuronedisease and as Lou Gehrig's disease. It is now well recognized thatprotein misfolding plays a central role in both familial and sporadicALS (Matus et al. 2013 Int. J. Cell Biol. ID674751http://dx.doi.org/10.1155/2013/674751). Saxena et al. (NatureNeuroscience 2009 Vol. 12 pp 627-636) demonstrated that Salubrinalextends the life span of a G93A-SOD1 transgenic mouse model of motorneuron disease. More recently, Jiang et al. (Neuroscience 2014)demonstrated that Guanabenz delays the onset of disease symptoms,extends lifespan, improves motor performance and attenuates motor neuronloss in the SOD1 G93A mouse model of ALS. Without to be bound by atheory, it is anticipated that compounds of the invention which areguanabenz derivative PPP1R15A inhibitors will ameliorate diseasemanifestations of ALS with the SOD1 mutation G93A. Therefore, thecompounds of formula (I) and (II) can be used to treat both familial andsporadic forms of ALS.

Examples of seipinopathies include, but are not limited toBerardinelli-Seip congenital lipodystrophy type 2 (BSCL2)-related motordisease, congenital generalized lipodystrophy (CGL), Silver syndrome,distal hereditary motor neuropathy type V (dHMN-V). The expression ofmutant forms of seipin in cultured cells activates the unfolded proteinresponse (UPR) pathway and induces ER stress-mediated cell death (Ito &Suzuki, 2009 Brain 132: 87-15). According to a preferred embodiment, theinvention relates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof for the use in treatingseipinopathy.

In another embodiment, the demyelination disorder referred therein ismultiple sclerosis and related disease such as Schilder's disease.According to a preferred embodiment, the invention relates to a PPP1R15Ainhibitor of formula (I) or (II) or a pharmaceutical acceptable saltthereof for the use in treating multiple sclerosis.

Cystic Fibrosis (CF)

Norez et al. (2008 Eur. J. Pharmacol. Vol. 592 pp 33-40) demonstratedthat Guanabenz activates Ca2+ dependent chloride currents in cysticfibrosis human airway epithelial cells. Without to be bound by a theory,it is anticipated that compounds of the invention which are guanabenzderivative PPP1R15A inhibitors will ameliorate disease manifestations ofcystic fibrosis. According to a preferred embodiment, the inventionrelates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof for the use in treating cysticfibrosis.

Retinal Diseases.

Recently published literature has provided evidences that the UPR isinvolved in the development of retinal degeneration: inherited retinaldegeneration such as retinal ciliopathies & retinitis pigmentosa,macular degeneration, retinopathy of premarurity, light-induced retinaldegeneration, retinal detachment, diabetic retinopathy and glaucoma (forreview Gorbatyuk et Gorbatyuk 2013—Retinal degeneration: Focus on theunfolded protein response, Molecular Vision Vol. 19 pp 1985-1998).Emerging evidence supports a role of ER stress in retinal apoptosis andcell death (Jing et al., 2012, Exp Diabetes Res, 2012, 589589).

Retinal ciliopathies are a group of rare genetic disorders originatingfrom a defect in the primary cilium of photoreceptors thus inducingretinitis pigmentosa. This defect has been reported to induce an ERstress due to protein accumulation in the inner segment of thephotoreceptor which in turn induces the UPR (WO2013/124484). Retinaldegeneration is a very common feature in ciliopathies that can beobserved either in isolated retinitis pigmentosa such as Leber'scongenital amaurosis or X-linked retinitis pigmentosa, or also insyndromic conditions like the Bardet-Biedl Syndrome (BBS), the Alströmsyndrome (ALMS) or Usher syndrome. The retinal ciliopathy is selectedfrom the group consisting of Bardet-Biedl syndrome, Senior-Lokensyndrome, Joubert syndrome, Salidono-Mainzer syndrome, Sensenbrennersyndrome, Jeune syndrome, Meckel-Gruder syndrome, Alström syndrome, MORMsyndrome, Leber's congenital amaurosis caused by mutation in a ciliarygene and X-linked retinitis pigmentosa caused by mutation in the RPGRgene.

Retinitis pigmentosa is an inherited, degenerative eye disease thatcauses severe vision impairment and often blindness. It is the mostcommon cause of genetically determined blindness. Sufferers willexperience one or more of the following symptoms: night blindness;tunnel vision (no peripheral vision); peripheral vision (no centralvision); latticework vision; aversion to glare; slow adjustment fromdark to light environments and vice versa; blurring of vision; poorcolor separation; and extreme tiredness. Retinitis pigmentosa (RP) iscaused by over 100 mutations in the rhodopsin gene (Dryja et al., 1991,Proc Natl Acad Sci USA, 88, 9370-4). Rhodopsin is a G protein-coupledreceptor that transduces light in the rod photoreceptors and consists ofa covalent complex between the transmembrane protein opsin of 348 aminoacids, covalently bound to 11-cis retinal (Palczewski, 2006, Annu RevBiochem, 75, 743-67). The RP-causing rhodopsin mutations are mostlymissense mutations distributed throughout the protein (Dryja et al.,1991), similar to the ALS-causing SOD1 mutations (Valentine et al.,2005, Annu Rev Biochem, 74, 563-93). The RP-causing rhodopsin mutantshave been studied in diverse systems and results from heterologousexpression of the proteins in mammalian cells, in transgenic mice anddrosophila are consistent (Griciuc et al., 2011, Trends Mol Med, 17,442-51). The most prevalent RP-causing rhodopsin mutants fail to fold,do not bind 11-cis-retinal, do not reach the cell surface but areretained in the ER (Griciuc et al., 2011, Trends Mol Med, 17, 442-51).Misfolding of the rhodopsin mutants causes ER stress and rod cell death(Griciuc et al., 2011). This strongly suggests that PPP1R15A inhibitorslike Guanabenz but which advantageously exhibits no activity toward theadrenergic alpha2A receptor, like compounds of the invention, willameliorate RP.

In one preferred embodiment, the compound of formula (I) or (II) is foruse in treating retinal diseases, more preferably, inherited retinaldegeneration such as retinal ciliopathies, retinitis pigmentosa, maculardegeneration, retinopathy of premarurity, light-induced retinaldegeneration, retinal detachment, diabetic retinopathy and glaucoma.According to a preferred embodiment, the invention relates to a PPP1R15Ainhibitor of formula (I) or (II) or a pharmaceutical acceptable saltthereof for the use in treating syndromic retinitis pigmentosa and/ornon-syndromic retinitis pigmentosa. According to a preferred embodiment,the invention relates to a PPP1R15A inhibitor of formula (I) or (II) ora pharmaceutical acceptable salt thereof for the use in treating Leber'scongenital amaurosis. According to another preferred embodiment, theinvention relates to a PPP1R15A inhibitor of formula (I) or apharmaceutical acceptable salt thereof for the use in treatingBardet-Biedl syndrome. According to another preferred embodiment, theinvention relates to a PPP1R15A inhibitor of formula (I) or apharmaceutical acceptable salt thereof for the use in treating Alströmsyndrome. According to another preferred embodiment, the inventionrelates to a PPP1R15A inhibitor of formula (I) or a pharmaceuticalacceptable salt thereof for the use in treating Usher syndrome.

Age-related macular degeneration (AMD) is the main cause of legalblindness among those over 65 years of age in the United States. Shen etal. (2011 Effect of Guanabenz on Rat AMD Models and Rabbit ChoroidalBlood—Vol. 5 pp 27-31) demonstrated that Guanabenz significantlyprotected retinal pigment epithelium (RPE) from NaIO3-induceddegeneration, inhibited the development of choroidal neovascularization(CNV) in laser-induced rat AMD model and increased choroidal blood flowmarkedly in vivo. Guanabenz derivative compounds of the invention whichare PPP1R15A inhibitors like Guanabenz but which advantageously exhibitno activity toward the adrenergic alpha2A receptor will be useful totreat retinal or macular degeneration.

In preferred embodiment, the compound of formula (I) is for use intreating retinal diseases, more preferably for use in treating diseasesselected in the group of inherited retinal degeneration such as retinalciliopathies, retinitis pigmentosa, macular degeneration, retinopathy ofpremarurity, light-induced retinal degeneration, retinal detachment,diabetic retinopathy and glaucoma in association with a compoundincreasing the expression and/or the activity of BIP protein, such asValproic acid or a derivative thereof, trichostatin A, lithium,1-(3,4-dihydroxy-penyl)-2-thiocyanate-ethanone and exendin-4. Thus, theinvention relates to composition comprising a PPP1R15A inhibitor offormula (I) or a pharmaceutical acceptable salt thereof and a compoundincreasing the expression and/or the activity of BIP protein, preferablyValproic acid, for use in the treatment of diseases selected in thegroup of inherited retinal degeneration such as retinal ciliopathies,retinitis pigmentosa, macular degeneration, retinopathy of premarurity,light-induced retinal degeneration, retinal detachment, diabeticretinopathy and glaucoma. In preferred embodiment, the compound offormula (I) or (II), is for use in treating retinal diseases, morepreferably for use in treating diseases selected in the group ofinherited retinal degeneration such as retinal ciliopathies, retinitispigmentosa, macular degeneration, retinopathy of premarurity,light-induced retinal degeneration, retinal detachment, diabeticretinopathy and glaucoma in association with a gene therapy vectors, Nonlimiting examples of gene therapy vectors include lentivirus,adenovirus, and adeno-associated vectors (AAVs); these vectors areeffective in delivering genes of interest to the retina and retinalpigment epithelium for ocular gene therapy. It is anticipated that in anocular gene therapy of inherited retinal degeneration associated with anaccumulation of mutated misfolded proteins, protein accumulation in theendoplasmic reticulum will remain present while a normal protein isexpressed from the gene therapy vector. It remains the need to decreasethe protein accumulation/load in the cell, preferably in the ER withPPP1R15A inhibitors. The invention also relates to compositioncomprising PPP1R15A inhibitor selected in the group of compound offormula (I) or (II), guanabenz and salubrinal or a pharmaceuticalacceptable salt thereof, in combination with ocular gene therapy.

Lysosomal Storage Diseases;

Lysosomal storage diseases are a group of approximately 50 rareinherited metabolic disorders that result from defects in lysosomalfunction. The lysosomal dysfunction is usually the consequence ofdeficiency of a single enzyme required for the metabolism of lipids,glycoproteins or so-called mucopolysaccharides. Examples of lysosomalstorage diseases which can be treated with by PPP1R15A inhibitors offormula (I) or (II) described herein include, but are not limited to,Activator Deficiency/GM2 gangliosidosis, alpha-mannosidosis,aspartylglucosaminuria, cholesteryl ester storage disease, cystinosis,Danon disease, Fabry disease, Farber disease, Niemann-Pick disease,fucosidosis, galactosialidosis, Gaucher disease (Types I, II, II), GM1gangliosidosis (infantile, late infantile/juvenile, adult/chronic),I-cell disease/Mucolipidosis, Infantile free sialic acid storagedisease/ISSD, Juvenile hexosaminidase A deficiency, Krabbe disease(infantile onset, late onset), lysosomal acid lipase deficiency (earlyonset/late onset), metachromatic leukodystrophy, mucopolysaccharidosesdisorders (such as Pseudo-Hurler polydystrophy/mucolipidosis IIIA,mucopolysaccharidosis I (MPS I) Hurler syndrome, MPS I Scheie syndrome,MPS I Hurler-Scheie syndrome, MPS II Hunter syndrome, Sanfilipposyndrome Type A (MPS IIIA), Sanfilippo syndrome Type B (MPS IIIB),Sanfilippo syndrome Type C (MPS IIIC), Sanfilippo syndrome Type D (MPSIIID), Morquio Type A/MPS IVA, Morquio Type B/MPS IVB, MPS IXhyaluronidase deficiency, MPS VI Maroteaux-Lamy, MPS VII Sly syndrome,mucopolylipidosis l/sialidosis, mucolipidosis IIIC, mucolipidosis typeIV (multiple sulfatase deficiency, Niemann-Pick disease (Types A, B, C),CLN6 disease (atypical late infantile, late onset variant, earlyjuvenile), Batten-Spielmeyer-Vogt/Juvenile NCUCLN3 disease, FinnishVariant late infantile CLN5, Jansky-Bielschosky disease/late infantileCLN2/TPP1 disease, Kufs/Adult-onset NCUCLN4 disease, Northernepilepsy/variant late infantile CLN8, Santavuori-Haltia/infantileCLN1/PPT disease, beta-mannosidosis, Pompe disease/glycogen storagedisease type II, pycnodysostosis, Sandhoff disease/GM2 gangliosidosis(adult onset, infantile onset, juvenile onset), Schindler disease, SalIdisease/sialic acid storage disease, Tay-Sachs/GM2 gangliosidosis, andWolman disease. According to preferred embodiment, the invention relatesto a PPP1R15A inhibitor of formula (I) or (II) or a pharmaceuticalacceptable salt thereof for the use in treating lysosomal storagediseases which are the consequence of deficiency of at least one singleenzyme required for the metabolism of lipids, glycoproteins or so-calledmuco-polysaccharides and wherein said enzyme is misfolded in theendoplasmic reticulum (ER). According to a preferred embodiment, thelysosomal storage disease is Gaucher disease.

Amyloidosis Diseases:

Amyloidosis is a non-specific term that refers to a number of differentdiseases collectively called amyloidoses. Amyloids are proteins whosesecondary structure change, causing the proteins to fold in acharacteristic form, the beta-pleated sheet. When the normally solubleproteins fold to become amyloids, they become insoluble, deposit andaccumulate in organs or tissues, disrupting normal function. Differenttypes of amyloidoses have different signs and symptoms depending onwhere and in which organs the amyloid proteins aggregate. Example ofamyloidosis diseases includes, but are not limited to, AL, AH, ALHamyloidosis (amyloid derived from light-chain, heavy-chain, heavy andlight chain antibodies respectively), AA amyloidosis (amyloid derivedfrom derived from serum A protein), ATTR amyloidosis (amyloid derivedfrom transthyrethin), primary systemic amyloidosis, secondary systemicamyloidosis, senile systemic amyloidodis, familial amyloidpolyneuropathy 1, hereditary cerebral amyloid angiopathy,hemodialysis-related amyloidosis, familial amyloid polyneuropathy III,Finnish hereditary systemic amyloidosis, atrial amyloidosis, hereditarynon-neuropathic systemic amyloidosis, injection-localized amyloidosisand hereditary renal amyloidosis and Alzheimer disease among others.

According to another preferred embodiment, the amyloid is Amyloid beta(Aβ or Abeta) and the invention relates to a PPP1R15A inhibitor offormula (I) or (II) or a pharmaceutical acceptable salt thereof for theuse in treating Alzheimer disease.

According to another preferred embodiment, the amyloid is HLA-B27(Colbert et al. 2009 Prion Vol. 3 (1) pp 15-16) and the inventionrelates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof for the use in treatingspondylo-arthropathies, more preferably ankylosing spondylitis.

Cancers

Cancer cells have high metabolic requirement and their proliferationrelies on efficient protein synthesis. Translation initiation plays acrucial role in controlling protein homeostasis, differentiation,proliferation and malignant transformation. Increasing translationinitiation contributes to cancer initiation and conversely, decreasingtranslation initiation could reduce tumor growth (Donze et al., 1995,EMBO J, 14, 3828-34; Pervin et al., 2008, Cancer Res, 68, 4862-74; Chenet al., 2011, Nat Chem Biol, 7, 610-6). Without wishing to be bound bytheory, it is believed that inhibiting PPP1R15A could selectively reducetranslation in tumor cells and thus reduce tumor growth. Examples oftypes of cancer which can be treated by PPP1R15A inhibitors of formula(I) or (II) disclosed herein include but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia. More particular examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,neuroblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, mammary cancer, colon cancer,colorectal cancer, endometrial carcinoma, salivary gland carcinoma,kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma, osteosarcoma, stomach cancer, melanoma,multiple myeloma, medullary carcinoma of the thyroid and head and neckcancer.

Inflammation

PPP1R15A represents a promising target to control inflammation byblocking the release of inflammatory cytokines and other secretedmolecular mediators leading to pathogenic conditions. Non-limitingexamples of diseases or conditions having inflammation associatedtherewith which can be treated with by PPP1R15A inhibitors of formula(I) or (II) described herein include, but are not limited toinfection-related or non-infectious inflammatory conditions in the lung(i.e., sepsis, lung infections, Respiratory Distress Syndrome,bronchopulmonary dysplasia, etc.); infection-related or non-infectiousinflammatory conditions in other organs such as colitis, ulcerativecolitis, Inflammatory Bowel Disease, diabetic nephropathy, hemorrhagicshock, spondylo-arthropathies, pancreatitis; inflammation-induced cancer(i.e., cancer progression in patients with colitis or Inflammatory BowelDisease); and the like. Examples of such pathogenic inflammatoryconditions include auto-immune diseases, hereditary diseases, chronicdiseases and infectious diseases such as allergy, asthma,hypercytokinemia including graft versus host disease (GVHD), acuterespiratory distress syndrome (ARDS), sepsis, systemic inflammatoryresponse syndrome (SIRS) (see WO2011/061340). Preferably, infectiousdisease is selected from influenza virus infection, smallpox virusinfection, herpes virus infection, severe acute respiratory syndrome(SARS), chikungunya virus infection, West Nile Virus infection, denguevirus infection, Japanese encephalitis virus infection, yellow fevervirus infection, and hepatitis C virus infection.

Preferably auto-immune disease is selected from Sjögren's syndrome,systemic lupus erythematosus, psoriasis, dermatitis herpetiformis,vitiligo, mycosis fungoides, allergic contact dermatitis, atopicdermatitis, lichen planus, Pityriasis lichenoides et varioliforms acuta(PLEVA), arthritis, catastrophic antiphospholipid syndrome.

According to another preferred embodiment, the invention relates to aPPP1R15A inhibitor of formula (I) or (II), or a pharmaceuticalacceptable salt thereof, for the use in treating a disease selected inthe group of colitis, ulcerative colitis, Inflammatory Bowel Disease,pancreatitis, sepsis. According to another preferred embodiment, theinvention relates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof, for the use in treatingpancreatitis. According to another preferred embodiment, the inventionrelates to a PPP1R15A inhibitor of formula (I) or (II) or apharmaceutical acceptable salt thereof, for the use in treating sepsis.According to another preferred embodiment, the invention relates to aPPP1R15A inhibitor of formula (I) or (II), or a pharmaceuticalacceptable salt thereof, for the use in treating spondylo-arthropathies,more preferably ankylosing spondylitis.

-   -   Metabolic and/or cardio-vascular disorders, such adiposity,        hyper-lipidemia, familial hyper-cholesterolemia, obesity,        atherosclerosis, hypertension, heart diseases, cardiac        ischaemia, stroke, myocardial infraction, trans-aortic        constriction, and diabetes and related disorders include        hyperglycemia, impaired glucose tolerance, hyper-insulinemia        (pre-diabetes), insulin hypersensitivity type I and II diabetes,        insulin resistance, Wolcott-Rallison Syndrome among others.

In one preferred embodiment, the compound of formula (I) or (II) is foruse in treating pre-diabetes or diabetes, more preferably type 2pre-diabetes or type 2 diabetes. In another preferred embodiment, thecompound of formula (I) or (II) is for use in treating a diseaseselected in the group of hyperglycemia, impaired glucose tolerance,hyper-insulinemia (pre-diabetes), insulin hypersensitivity type I andII, insulin resistance and Wolcott-Rallison Syndrome. Indeed, theinsulin-secreting β-cells in the pancreas have a heavy and tightlyregulated biosynthetic burden consisting in insulin secretion. Thus,these cells have an important need to maintain ER homeostasis (Back andKaufman, 2012, Annu Rev Biochem, 81, 767-93). Type 2 diabetes ismanifested by increased levels of blood glucose due to insulinresistance in the adipose, muscle and liver and/or impaired insulinsecretion from pancreatic β-cells. As a response, β-cells mass increaseand their function is enhanced. Eventually, the burden on the β-cells istoo high leading to their progressive decline and death. Increasingevidence reveals that death of β-cells results from ER stress (Back andKaufman, 2012, Annu Rev Biochem, 81, 767-93). Importantly, Chop deletionimproves β-cells function in diverse models of diabetes (Song et al.,2008, J Clin Invest, 118, 3378-89). Without wishing to be bound bytheory, it is believed that inhibitors of PPP1R15A-PP1 will improveβ-cells function in type 2 diabetes since inhibition of PPP1R15A-PP1reduces the levels of the pro-apoptotic protein CHOP during ER stress(Tsaytler et al., 2011, Science).

In another embodiment, the compound of formula (I) or (II) is for use intreating a disease selected in the group of hypertension, heartdiseases, cardiac ischaemia, stroke, myocardial infraction, trans-aorticconstriction or vascular stroke. In another preferred embodiment, thecompound of formula (I) or (II) is for use in treating cardiac ischemia.In another preferred embodiment, the compound of formula (I) or (II) isfor use in treating atherosclerosis.

Osteoporosis:

Yokota et al. (BMC Musculoskeletal disorders 2013, 14, 197) and He etal. (Cellular Signaling 2013, 25 552-560) demonstrated that Salubrinal(Boyce et al. 2005) efficiently block osteoporosis in mice model andstimulates bone formation. However, Salubrinal is toxic and cannot beused to treat human patients. In contrast, the PPP1R15A inhibitors offormula (I) or (II) are predicted to be safe and could be useful for thetreatment of osteoporosis. The compound of formula (I) or (II) is foruse in treating osteoporosis.

Central Nervous System Trauma

Ohri et al. (Neurobiology of disease, 2013 Vol. 58 pp 29-37)demonstrated that Salubrinal significantly improved hindlimb locomotionwhich corresponds with an improved white matter sparing and a decreasedoligodendrocytes apoptosis, thus improving functional recovery afterspinal cord injury. Therefore, the PPP1R15A inhibitors of formula (I) or(II) of the invention are predicted to be safe and could be useful toreduce the oligodendrocytes loss after traumatic spinal cord injury andfor the treatment of spinal cord injury. In one preferred embodiment,the compound of formula (I) or (II) is for the prophylactic and/ortherapeutic treatment of spinal cord injury.

Ischemia, Cerebral Ischemia, Sleep Apnoea

The present invention provides methods of using PPP1R15A inhibitors offormula (I) or (II) of the invention to prevent and/or treat tissuedamage resulting from cell damage or death due to necrosis or apoptosis.Example of neural tissue damage include ischemia and reperfusion injury,such as cerebral ischemic stroke and head trauma. In one preferredembodiment, the compound of formula (I) or (II) is for the prophylacticand/or therapeutic treatment of cerebral ischemia, such as cerebralischemic stroke and head trauma.

Aging

Aging is associated with the degeneration of cells, tissues, and organs,resulting in diseases such as cancer, cardiovascular failure, obesity,type 2 diabetes mellitus, non-alcoholic fatty liver, andneurodegenerative diseases, as well as the decline of most measures ofphysiological performance.

In biology, senescence is the state or process of aging. Cellularsenescence is a phenomenon where isolated cells demonstrate a limitedability to divide in culture (the Hayflick Limit, discovered by LeonardHayflick in 1961), while organismal senescence is the ageing oforganisms. Organismal senescence is characterised by the decliningability to respond to stress, increasing homeostatic imbalance and theincreased risk of disease; in particular, the UPR is impaired with age(Naidoo et al., 2008, J Neurosci, 28, 6539-48). Thus, prolonging thebeneficial effect of the UPR by inhibition of elF2α phosphatase couldameliorate age-related disorders. Therefore, the PPP1R15A inhibitors offormula (I) or (II) of the invention are predicted to be safe and couldbe useful to prevent and/or treat diseases or disorders relating tolifespan or proliferative capacity of cells, and diseases or diseaseconditions induced or exacerbated by cellular senescence in an animal,more specifically humans.

According to an embodiment, the present invention also concernscompounds of formula (I) or (II) for use in the treatment and/orprevention of a disorder selected in the group of tauopathies chosenfrom Alzheimer disease, progressive supranuclear palsy, corticobasaldegeneration, frontotemporal lobar degeneration or frontotemporaldementia (FTD) (Pick's disease); synucleinopathies chosen fromParkinson's disease, dementia with Lewy bodies, pure autonomic failure,and multiple system atrophy; polyglutamine and polyalanine diseaseschosen from Huntington disease, spinobulbar muscular atrophy (or Kennedydisease), dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxiatype 1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3 (orMachado-Joseph disease), Spinocerebellar ataxia type 6, Spinocerebellarataxia type 7 and Spinocerebellar ataxia type 17, oculo-pharyngealmuscular dystrophy; demyelinating disorders like leukodystrophies,Charcot-Marie-Tooth disease and multiple sclerosis, cystic fibrosis,seipinopathies, lysosomal storage disorders, amyloidosis diseases,inflammation, metabolic disorders and cardio-vascular disorders chosenfrom adiposity, hyper-lipidemia, familial hyper-cholesterolemia,obesity, atherosclerosis, hypertension, heart diseases, cardiacischaemia, stroke, myocardial infraction, trans-aortic constriction,vascular stroke; osteoporosis, nervous system trauma, ischemia,osteoporosis, retinal diseases, like retinitis pigmentosa, retinalciliopathies, glaucoma, macular degeneration and aging.

According to an embodiment, the disorder is more particularly selectedfrom multiple sclerosis; a leukodystrophy, preferablyPelizaeus-Merzbacher disease; a demyelinating disorder such asCharcot-Marie-Tooth, preferably CMT-1A; a cardio-vascular disorder suchas hypertension, heart diseases, cardiac ischaemia, stroke, myocardialinfraction, trans-aortic constriction; colitis, ulcerative colitis,Inflammatory Bowel Disease, pancreatitis, sepsis; an amyloidosisdisease, such as Alzheimer disease and ankylosing spondylitis;pre-diabetes and diabetes, such as type -2 diabetes.

According to a further embodiment, the present invention also concerns acompound of formula (I) or (II) in association with a compoundincreasing the expression and/or the activity of BIP protein, for use intreating retinal diseases selected in the group of inherited retinaldegeneration such as retinal ciliopathies, retinitis pigmentosa, maculardegeneration, retinopathy of premarurity, light-induced retinaldegeneration, retinal detachment, diabetic retinopathy and glaucoma.

Pharmaceutical Compositions

For use according to the present invention, the compounds orphysiologically acceptable salts, esters or other physiologicallyfunctional derivatives thereof, described herein, may be presented as apharmaceutical formulation, comprising the compounds or physiologicallyacceptable salt, ester or other physiologically functional derivativethereof, together with one or more pharmaceutically acceptable carrierstherefore and optionally other therapeutic and/or prophylacticingredients. The carrier(s) must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. The pharmaceutical compositionsmay be for human or animal usage in human and veterinary medicine.Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in the“Handbook of Pharmaceutical Excipients, 2^(nd) Edition, (1994), Editedby A Wade and PJ Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water. The choice ofpharmaceutical carrier, excipient or diluent can be selected with regardto the intended route of administration and standard pharmaceuticalpractice. The pharmaceutical compositions may comprise as, or inaddition to, the carrier, excipient or diluent any suitable binder(s),lubricant(s), suspending agent(s), coating agent(s), solubilisingagent(s), buffer(s), flavouring agent(s), surface active agent(s),thickener(s), preservative(s) (including anti-oxidants) and the like,and substances included for the purpose of rendering the formulationisotonic with the blood of the intended recipient.

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal, ocular and sublingual), rectal or parenteral(including subcutaneous, intradermal, intramuscular and intravenous),nasal, intra-ocularly and pulmonary administration e.g., by inhalation.The formulation may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods include the step of bringinginto association an active compound with liquid carriers or finelydivided solid carriers or both and then, if necessary, shaping theproduct into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules or tablets each containing apredetermined amount of active compound. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine an active compound in a free-flowing form such as apowder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating agent, surface-active agent or dispersing agent.Moulded tablets may be made by moulding an active compound with an inertliquid diluent. Tablets may be optionally coated and, if uncoated, mayoptionally be scored. Capsules may be prepared by filling an activecompound, either alone or in admixture with one or more accessoryingredients, into the capsule shells and then sealing them in the usualmanner. Cachets are analogous to capsules wherein an active compoundtogether with any accessory ingredient(s) is sealed in a rice paperenvelope. An active compound may also be formulated as dispersiblegranules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged,e.g., in a sachet. Formulations suitable for oral administration whereinthe carrier is a liquid may be presented as a solution or a suspensionin an aqueous or non-aqueous liquid, or as an oil-in-water liquidemulsion.

Formulations for oral administration include controlled release dosageforms, e.g., tablets wherein an active compound is formulated in anappropriate release—controlling matrix, or is coated with a suitablerelease—controlling film. Such formulations may be particularlyconvenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of an active compound with the softenedor melted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of an active compound inaqueous or oleaginous vehicles.

Pharmaceutical formulations of the invention are suitable for ophthalmicadministration, in particular for intra-ocular, topical ocular orperi-ocular administration, more preferably for topical ocular orperi-ocular administration.

Injectible preparations may be adapted for bolus injection or continuousinfusion. Such preparations are conveniently presented in unit dose ormulti-dose containers which are sealed after introduction of theformulation until required for use. Alternatively, an active compoundmay be in powder form which is constituted with a suitable vehicle, suchas sterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depotpreparations, which may be administered by intramuscular injection or byimplantation, e.g., subcutaneously or intramuscularly. Depotpreparations may include, for example, suitable polymeric or hydrophobicmaterials, or ion-exchange resins. Such long-acting formulations areparticularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavityare presented such that particles containing an active compound anddesirably having a diameter in the range of 0.5 to 7 microns aredelivered in the bronchial tree of the recipient. As one possibilitysuch formulations are in the form of finely comminuted powders which mayconveniently be presented either in a pierceable capsule, suitably of,for example, gelatin, for use in an inhalation device, or alternativelyas a self-propelling formulation comprising an active compound, asuitable liquid or gaseous propellant and optionally other ingredientssuch as a surfactant and/or a solid diluent. Suitable liquid propellantsinclude propane and the chlorofluorocarbons, and suitable gaseouspropellants include carbon dioxide. Self-propelling formulations mayalso be employed wherein an active compound is dispensed in the form ofdroplets of solution or suspension.

Such self-propelling formulations are analogous to those known in theart and may be prepared by established procedures. Suitably they arepresented in a container provided with either a manually-operable orautomatically functioning valve having the desired spraycharacteristics; advantageously the valve is of a metered typedelivering a fixed volume, for example, 25 to 100 microlitres, upon eachoperation thereof.

As a further possibility an active compound may be in the form of asolution or suspension for use in an atomizer or nebuliser whereby anaccelerated airstream or ultrasonic agitation is employed to produce afine droplet mist for inhalation.

Formulations suitable for nasal administration include preparationsgenerally similar to those described above for pulmonary administration.When dispensed such formulations should desirably have a particlediameter in the range 10 to 200 microns to enable retention in the nasalcavity; this may be achieved by, as appropriate, use of a powder of asuitable particle size or choice of an appropriate valve. Other suitableformulations include coarse powders having a particle diameter in therange 20 to 500 microns, for administration by rapid inhalation throughthe nasal passage from a container held close up to the nose, and nasaldrops comprising 0.2 to 5% w/v of an active compound in aqueous or oilysolution or suspension.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Additionally, such pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, inert gases and the like.

Formulations suitable for topical formulation may be provided forexample as gels, creams or ointments. Such preparations may be appliede.g. to a wound or ulcer either directly spread upon the surface of thewound or ulcer or carried on a suitable support such as a bandage,gauze, mesh or the like which may be applied to and over the area to betreated.

Liquid or powder formulations may also be provided which can be sprayedor sprinkled directly onto the site to be treated, e.g. a wound orulcer. Alternatively, a carrier such as a bandage, gauze, mesh or thelike can be sprayed or sprinkle with the formulation and then applied tothe site to be treated.

According to a further aspect of the invention, there is provided aprocess for the preparation of a pharmaceutical or veterinarycomposition as described above, the process comprising bringing theactive compound(s) into association with the carrier, for example byadmixture.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active agent with liquid carriers orfinely divided solid carriers or both, and then if necessary shaping theproduct. The invention extends to methods for preparing a pharmaceuticalcomposition comprising bringing a compound of general formula (I) inconjunction or association with a pharmaceutically or veterinarilyacceptable carrier or vehicle.

Salts

The compounds of the invention can be present as salts, in particularpharmaceutically and veterinarily acceptable salts.

Pharmaceutically acceptable salts of the compounds of the inventioninclude suitable acid addition or base salts thereof. A review ofsuitable pharmaceutical salts may be found in Berge et al, J Pharm Sci,66, 1-19 (1977). Salts are formed, for example with strong inorganicacids such as mineral acids, e.g. hydrohalic acids such ashydrochloride, hydrobromide and hydroiodide, sulfuric acid, phosphoricacid sulphate, bisulphate, hemisulphate, thiocyanate, persulphate andsulphonic acids; with strong organic carboxylic acids, such asalkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted orsubstituted (e.g., by halogen), such as acetic acid; with saturated orunsaturated dicarboxylic acids, for example oxalic, malonic, succinic,maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylicacids, for example ascorbic, glycolic, lactic, malic, tartaric or citricacid; with aminoacids, for example aspartic or glutamic acid; withbenzoic acid; or with organic sulfonic acids, such as (C₁-C₄)-alkyl- oraryl-sulfonic acids which are unsubstituted or substituted (for example,by a halogen) such as methane- or p-toluene sulfonic acid. Salts whichare not pharmaceutically or veterinarily acceptable may still bevaluable as intermediates.

Preferred salts include, for example, acetate, trifluoroacetate,lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate,adipate, alginate, aspartate, benzoate, butyrate, digluconate,cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate,hexanoate, fumarate, nicotinate, palmoate, pectinate,3-phenylpropionate, picrate, pivalate, proprionate, tartrate,lactobionate, pivolate, camphorate, undecanoate and succinate, organicsulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids. According to a preferred embodiment the salt isacetate.

Enantiomers/Tautomers

In all aspects of the present invention previously discussed, theinvention includes, where appropriate all enantiomers, diastereoisomersand tautomers of the compounds of the invention. The person skilled inthe art will recognise compounds that possess optical properties (one ormore chiral carbon atoms) and/or tautomeric characteristics. Thecorresponding enantiomers and/or tautomers may be isolated/prepared bymethods known in the art. Enantiomers are characterised by the absoluteconfiguration of their chiral centres and described by the R- andS-sequencing rules of Cahn, Ingold and Prelog. Such conventions are wellknown in the art (e.g. see ‘Advanced Organic Chemistry’, 3^(rd) edition,ed. March, J., John Wiley and Sons, New York, 1985).

Compounds of formula (I) or (II) thus also include the tautomer forms offormula:

As an illustrative example, a tautomer form of Compound 2 is:

Compounds of the invention containing a chiral centre may be used as aracemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone.

Stereo and Geometric Isomers

Some of the compounds of the invention may exist as stereoisomers and/orgeometric isomers—e.g. they may possess one or more asymmetric and/orgeometric centres and so may exist in two or more stereoisomeric and/orgeometric forms as E/Z (Entgegen/Zusammen) isomers. The presentinvention contemplates the use of all the individual stereoisomers andgeometric isomers of those inhibitor agents, and mixtures thereof. Theterms used in the claims encompass these forms, provided said formsretain the appropriate functional activity (though not necessarily tothe same degree).

Compounds of formula (I) or (II) thus also include the E and/or Z isomerforms of formula:

The present invention also includes all suitable isotopic variations ofthe agent or a pharmaceutically acceptable salt thereof. An isotopicvariation of an agent of the present invention or a pharmaceuticallyacceptable salt thereof is defined as one in which at least one atom isreplaced by an atom having the same atomic number but an atomic massdifferent from the atomic mass usually found in nature. Examples ofisotopes that can be incorporated into the agent and pharmaceuticallyacceptable salts thereof include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorus, sulfur, fluorine and chlorine such as ²H, ³H, ¹³C,¹⁴C, ¹⁶N, ¹⁷O, ¹⁸O, ³¹F, ³²F, ³⁶S, ¹⁸F and ³⁶Cl, respectively. Certainisotopic variations of the agent and pharmaceutically acceptable saltsthereof, for example, those in which a radioactive isotope such as ³H or¹⁴C is incorporated, are useful in drug and/or substrate tissuedistribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with isotopes such as deuterium,i.e., ²H, may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example, increased in vivo half-life orreduced dosage requirements and hence may be preferred in somecircumstances. For example, the invention includes compounds of generalformula (I) where any hydrogen atom has been replaced by a deuteriumatom. Isotopic variations of the agent of the present invention andpharmaceutically acceptable salts thereof of this invention cangenerally be prepared by conventional procedures using appropriateisotopic variations of suitable reagents.

Prodrugs

The invention further includes the compounds of the present invention inprodrug form, i.e. covalently bonded compounds which release the activeparent drug according to general formula (I) in vivo. Such prodrugs aregenerally compounds of the invention wherein one or more appropriategroups have been modified such that the modification may be reversedupon administration to a human or mammalian subject. Reversion isusually performed by an enzyme naturally present in such subject, thoughit is possible for a second agent to be administered together with sucha prodrug in order to perform the reversion in vivo. Examples of suchmodifications include ester (for example, any of those described above),wherein the reversion may be carried out be an esterase etc. Other suchsystems will be well known to those skilled in the art.

Solvates

The present invention also includes solvate forms of the compounds ofthe present invention. The terms used in the claims encompass theseforms.

Polymorphs

The invention further relates to the compounds of the present inventionin their various crystalline forms, polymorphic forms and (an)hydrousforms. It is well established within the pharmaceutical industry thatchemical compounds may be isolated in any of such forms by slightlyvarying the method of purification and or isolation form the solventsused in the synthetic preparation of such compounds.

Administration

The pharmaceutical compositions of the present invention may be adaptedfor rectal, nasal, intrabronchial, topical (including buccal, sublingualand ophthalmic administration, in particular for intra-ocular,intra-vitreal, topical ocular or peri-ocular administration), vaginal orparenteral (including subcutaneous, intramuscular, intravenous,intraarterial and intradermal), intraperitoneal or intrathecaladministration. Preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, i.e., in the form of discrete portions containing a unitdose, or a multiple or sub-unit of a unit dose. By way of example, theformulations may be in the form of tablets and sustained releasecapsules, and may be prepared by any method well known in the art ofpharmacy.

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, gellules, drops, cachets,pills or tablets each containing a predetermined amount of the activeagent; as a powder or granules; as a solution, emulsion or a suspensionof the active agent in an aqueous liquid or a non-aqueous liquid; or asan oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or asa bolus etc. Preferably, these compositions contain from 1 to 250 mg andmore preferably from 10-100 mg, and even more preferably from 1-100 mg,of active ingredient per dose.

For compositions for oral administration (e.g. tablets and capsules),the term “acceptable carrier” includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally, intra-ocularly,topical, peri-ocularly or intramuscularly, and which are prepared fromsterile or sterilisable solutions.

The pharmaceutical compositions of the present invention may also be inform of suppositories, pessaries, suspensions, emulsions, lotions,ointments, creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific compound employed, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

In accordance with this invention, an effective amount of a compound ofgeneral formula (I) may be administered to target a particular conditionor disease. Of course, this dosage amount will further be modifiedaccording to the type of administration of the compound. For example, toachieve an “effective amount” for acute therapy, parenteraladministration of a compound of general formula (I) is preferred. Anintravenous infusion of the compound in 5% dextrose in water or normalsaline, or a similar formulation with suitable excipients, is mosteffective, although an intramuscular bolus injection is also useful.Typically, the parenteral dose will be about 0.01 to about 100 mg/kg;preferably between 0.1 and 20 mg/kg, in a manner to maintain theconcentration of drug in the plasma at an effective concentration Thecompounds may be administered one to four times daily at a level toachieve a total daily dose of about 0.4 to about 400 mg/kg/day. Theprecise amount of an inventive compound which is therapeuticallyeffective, and the route by which such compound is best administered, isreadily determined by one of ordinary skill in the art by comparing theblood level of the agent to the concentration required to have atherapeutic effect.

The compounds of this invention may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto achieve one or more of the therapeutic indications disclosed herein.Typically, a pharmaceutical composition containing the compound isadministered at an oral dose of between about 0.1 to about 50 mg/kg in amanner consistent with the condition of the patient. Preferably the oraldose would be about 0.1 to about 20 mg/kg.

No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention. The compounds of this invention, which may have goodbioavailability, may be tested in one of several biological assays todetermine the concentration of a compound which is required to have agiven pharmacological effect.

Combinations

In a particularly preferred embodiment, the one or more compounds of theinvention are administered in combination with one or more other activeagents, for example, existing drugs available on the market. In suchcases, the compounds of the invention may be administered consecutively,simultaneously or sequentially with the one or more other active agents.

Drugs in general are more effective when used in combination. Inparticular, combination therapy is desirable in order to avoid anoverlap of major toxicities, mechanism of action and resistancemechanism(s). Furthermore, it is also desirable to administer most drugsat their maximum tolerated doses with minimum time intervals betweensuch doses. The major advantages of combining drugs are that it maypromote additive or possible synergistic effects through biochemicalinteractions and also may decrease the emergence of resistance.

Beneficial combinations may be suggested by studying the inhibitoryactivity of the test compounds with agents known or suspected of beingvaluable in the treatment of a particular disorder. This procedure canalso be used to determine the order of administration of the agents,i.e. before, simultaneously, or after delivery. Such scheduling may be afeature of all the active agents identified herein.

According to preferred embodiment, the invention relates to apharmaceutical composition comprising a PPP1R15A inhibitor of formula(I) or (II), or a pharmaceutical acceptable salt thereof, and a compoundincreasing the expression and/or the activity of protein BiP and apharmaceutically acceptable carrier and/or excipient (seeWO2013/124484). Preferably, the compound increasing the expressionand/or activity of protein BiP is selected from the group consisting ofvalproic acid or a derivative thereof, trichostatin A, lithium,I-(3,4-dihydroxy-phenyl)-2-thiocyanate-ethanone, and exendin-4.According to a preferred embodiment the protein BiP is valproic acid ora derivative thereof such as 2-ene-valproic acid.

According to a preferred embodiment, the invention relates to apharmaceutical composition comprising a PPP1R15A inhibitor of formula(I) or (II), or a pharmaceutical acceptable salt thereof, and a compoundincreasing the expression and/or the activity of protein BiP and apharmaceutically acceptable carrier and/or excipient, to treat adisorder associated with the PPP1R15A pathway and associated withprotein misfolding stress and in particular with accumulation ofmisfolded proteins. Preferably, the disease is selected in the group ofcystic fibrosis, lysosomal storage disease, amyloidosis diseases,cancers, inflammation, metabolic disorders, cardio-vascular disorders,osteoporosis, central nervous system trauma, ischemia, retinal diseases,seipinopathies, tauopathies, synucleinopathies, polyglutamine andpolyalanine diseases, neurodegenerative diseases, preferably Alzheimer'sdisease, Parkinson's disease, Amyotrophic Lateral Sclerosis,Huntington's disease, Charcot Marie Tooth diseases, leukodystrophies,multiple sclerosis.

Assay

A further aspect of the invention relates to the use of a compound asdescribed above in an assay for identifying further candidate compoundscapable of inhibiting PPP1R15A-PP1. Preferably, the assay is acompetitive binding assay.

More preferably, the competitive binding assay comprises contacting acompound of the invention with PPP1R15A-PP1 and a candidate compound anddetecting any change in the interaction between the compound accordingto the invention and the PPP1R15A-PP1.

Preferably, the candidate compound is generated by conventional SARmodification of a compound of the invention. As used herein, the term“conventional SAR modification” refers to standard methods known in theart for varying a given compound by way of chemical derivatisation.

Thus, in one aspect, the identified compound may act as a model (forexample, a template) for the development of other compounds. Thecompounds employed in such a test may be free in solution, affixed to asolid support, borne on a cell surface, or located intracellularly. Theabolition of activity or the formation of binding complexes between thecompound and the agent being tested may be measured.

The assay of the present invention may be a screen, whereby a number ofagents are tested. In one aspect, the assay method of the presentinvention is a high through-put screen.

This invention also contemplates the use of competitive drug screeningassays in which neutralising antibodies capable of binding a compoundspecifically compete with a test compound for binding to a compound.

Another technique for screening provides for high throughput screening(HTS) of agents having suitable binding affinity to the substances andis based upon the method described in detail in WO 84/03564.

It is expected that the assay methods of the present invention will besuitable for both small and large-scale screening of test compounds aswell as in quantitative assays.

Preferably, the competitive binding assay comprises contacting acompound of the invention with PPP1R15A-PP1 in the presence of a knownsubstrate of PPP1R15A-PP1 and detecting any change in the interactionbetween said PPP1R15A-PP1 and said known substrate.

A further aspect of the invention provides a method of detecting thebinding of a ligand to PPP1R15A-PP1, said method comprising the stepsof:

-   (i) contacting a ligand with PPP1R15A-PP1 in the presence of a known    substrate-   (ii) detecting any change in the interaction between PPP1R15A-PP1    and said known substrate;-   and wherein said ligand is a compound of the invention.

One aspect of the invention relates to a process comprising the stepsof:

-   (a) performing an assay method described hereinabove;-   (b) identifying one or more ligands capable of binding to a ligand    binding domain; and-   (c) preparing a quantity of said one or more ligands.

Another aspect of the invention provides a process comprising the stepsof:

-   (a) performing an assay method described hereinabove;-   (b) identifying one or more ligands capable of binding to a ligand    binding domain; and-   (c) preparing a pharmaceutical composition comprising said one or    more ligands.

Another aspect of the invention provides a process comprising the stepsof:

-   (a) performing an assay method described hereinabove;-   (b) identifying one or more ligands capable of binding to a ligand    binding domain;-   (c) modifying said one or more ligands capable of binding to a    ligand binding domain;-   (d) performing the assay method described hereinabove;-   (e) optionally preparing a pharmaceutical composition comprising    said one or more ligands.

The invention also relates to a ligand identified by the methoddescribed hereinabove. Yet another aspect of the invention relates to apharmaceutical composition comprising a ligand identified by the methoddescribed hereinabove. Another aspect of the invention relates to theuse of a ligand identified by the method described hereinabove in thepreparation of a pharmaceutical composition for use in the treatment ofa disorder associated with accumulation of misfolded and/or unfoldedproteins as defined above.

The above methods may be used to screen for a ligand useful as aninhibitor of PPP1R15A-PP1.

Compounds of general formula (I) are useful both as laboratory tools andas therapeutic agents. In the laboratory certain compounds of theinvention are useful in establishing whether a known or newly discoveredtarget contributes a critical or at least significant biochemicalfunction during the establishment or progression of a disease state, aprocess commonly referred to as ‘target validation’.

The present invention is further described with reference to thefollowing figures wherein:

FIG. 1 shows dose dependent protection of Hela cells by compound 12 ofthe invention from ER stress induced by 6 hour exposure to tunicamycin.

FIG. 2 shows dose dependent protection of interferon-gamma injured ratoligodendrocytes by compound 11, compound 12 and compound 17 of theinvention.

FIG. 3 shows dose dependent protection of rotenone injured primarymesencephalic rat neurons by compound 5, compound 12 and compound 17 ofthe invention.

FIG. 4 shows dose dependent protection of amyloid-beta 1-42 injuredprimary cortical rat neurons by compound 12 of the invention.

FIG. 5 shows the ability of compound 12 and compound 17 at 5 microM and10 microM respectively to prevent the accumulation of T181P mutated DM20protein in Human 293T cell.

FIG. 6 shows the ability of compounds 16 to prevent cell deathassociated with the accumulation of misfold prone Insulin Akitaexpressed in Min6 cells.

FIG. 7 shows the ability of compound 12, compound 16 and compound 17 atdifferent concentrations to prevent Min6 insulinoma cell deathassociated with accumulation of misfolded protein induced by 6 hourexposure to tunicamycin.

FIG. 8 shows the ability of compound 11, compound 12, compound 16 andcompound 17 at different concentrations to prevent INS1 insulinoma celldeath associated with accumulation of misfolded protein induced by 6hour exposure to tunicamycin.

FIG. 9 shows the ability of compounds 6, 10, 11, 12, 15, 16 and 17 (at25 microM) to prevent type-I interferon production by mouse embryonicfibroblasts lipofected with poly I:C.

FIG. 10 shows the ability of compound 10 to protect neonatal ratcardiomyocytes against hypoxia-induced apoptosis. The graph shows thepercentage of apoptotic cells measured by FACS analysis. Cardiomyocyteswere exposed to hypoxia (0.3% O₂) for 36 h in the absence (0 μM) or inthe presence of indicated concentrations of Compound 2 (n=3).

The present invention is further described with reference to thefollowing non-limiting examples.

EXAMPLES

1—Methods & Materials

1.1—Preparation of the Compounds According to the Present Invention

The reactants and commercials compounds were purchased from AcrosOrganics, Sigma-Aldrich. The compounds according to the presentinvention can be prepared according to the following general procedure:

Compounds 1 & 2: Preparation of2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide FormateSalt (compound 1) and2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide (compound2) 2-(3-methylbutoxy)-1H-isoindole-1,3(2H)-dione (I-1)

Triethylamine (49.58 g) was added drop wise to a stirred solution ofN-Hydroxyphthalimide (40 g) and 1-bromo-3-methyl butane (37.4 g) in DMF(600 ml) at room temperature. The reaction mixture was stirred at 70° C.for 18 hours. The reaction mixture was allowed to cool to roomtemperature. The mixture was concentrated under reduced pressure and theresidue thus obtained was suspended in cold water (1000 ml). Theresulting suspension was stirred well for some time and the solid wasfiltered off under reduced pressure. The solid was further washed withdemineralized water (200 ml) and hexane (100 ml). The resulting solidwas dried under reduced pressure to get a crude material which waspurified by column chromatography using silica gel. The desired producteluted at around 2% Methanol in dichloromethane. Evaporation of pureproduct fractions gave 50.0 g of2-(3-methylbutoxy)-1H-isoindole-1,3(2H)-dione (Yield: 87.4%). ¹H-NMR(DMSO-d6): δ (ppm) 0.93 (d, 6H), 1.57 (q, 2H), 1.82 (m, 1H), 4.16 (t,2H), 7.86 (s, 4H); LC-MS: m/z=234.25 (M+H).

1-(amino-oxy)-3-methylbutane hydrochloride (I-2)

Hydrazine hydrate (12.8 g) was added drop-wise to a stirred solution of2-(3-methylbutoxy)-1H-isoindole-1,3(2H)-dione (45 g) in methanol (600ml) at room temperature. The reaction mixture was stirred at the sametemperature for 24 hours. The reaction mixture was filtered off toremove the insoluble by-product and the resulting filtrate wasconcentrated under reduced pressure to get a crude material which waspurified by column chromatography using silica gel. The desired producteluted at around 1% Methanol in dichloromethane. Evaporation of pureproduct fractions gave the desired intermediate as free base which wasconverted as hydrochloride salt using 4M HCl in 1,4-dioxane, to get 3.3g of 1-(aminooxy)-3-methylbutane hydrochloride. ¹H-NMR (DMSO-d6): δ(ppm) 0.89 (d, 6H), 1.46 (q, 2H), 1.65 (m, 1H), 4.01 (t, 2H), 10.84 (s,3H).

N′-(3-methylbutoxy)hydrazinecarboximidamide (I-3)

2N NaOH solution (3.6 ml) was added drop wise to a stirred solution of1-(amino-oxy)-3-methylbutane hydrochloride (1.2 g) ands-methylisothiosemicarbazide hydro-iodide (2.02 g) in water (3.6 ml) atroom temperature and was stirred for 48 hours. Then, the reactionmixtures was concentrated under reduced pressure and the residue wasazeotroped with methanol (5 ml). The resulting residue was suspended inethanol (10 ml) and insoluble inorganic salts were removed byfiltration. The filtrate was directly used for the next step without anyfurther processing. N′-(3-methylbutoxy)hydrazinecarboximidamide wasconfirmed by LCMS analysis. LC-MS: m/z=161.5 (M+H).

2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide formatesalt (compound 1)

2-chlorobenzaldehyde (1.81 g) was added drop wise to the filtrate whichcontain N′-(3-methylbutoxy)hydrazinecarboximidamide at room temperatureand was stirred for 2 hours. Then, the reaction mixture was concentratedunder reduced pressure and the residue thus obtained was furtherpurified by Prep HPLC using 0.1% HCOOH/water/MeCN to give 0.27 g of2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide asformate salt (Yield: 13.1%). ¹H-NMR (DMSO-d6): δ (ppm) 0.88 (d, 6H),1.48 (q, 2H), 1.68 (m, 1H), 3.75 (t, 2H), 7.32 (m, 2H), 7.44 (m, 2H),8.10 (m, 1H), 8.14 (m, 1H), 8.25 (m, 1H), 11.80 (s broad, 2H). LC-MS:m/z=282.88 (M+H).

2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide (compound2)

2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide formatesalt (220 mg) was dissolved in water and was basified by saturatedNaHCO₃ aqueous solution. The basic aqueous solution was extracted withDichloromethane and the organic layer was washed with water, dried oversodium sulphate and evaporated under reduced pressure to give 180 mg of2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide as freebase (Yield: 95%). ¹H-NMR (DMSO-d6): δ (ppm) 0.89 (d, 6H), 1.49 (q, 2H),1.69 (m, 1H), 3.75 (t, 2H), 5.73 (s broad, 2H), 7.30 (m, 2H), 7.44 (m,1H), 8.11 (m, 1H), 8.15 (m, 1H), 10.48 (s broad, 1H). LC-MS:

m/z=282.82 (M+H).

Compound 3: Preparation of2-(2-chlorobenzylidene)-N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide2-[2-(methylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (I-4)

2-chloroethyl methyl sulfide (10.1 g) was added drop-wise to a stirredsolution of N-Hydroxyphthalimide (12.5 g), potassium iodide (2.5 g) andpotassium carbonate (21.1 g) in DMF (150 ml) at room temperature and wasstirred at the 80° C. for 18 hours. The reaction mixture was allowed tocool to room temperature and was dumped in 500 ml of cold water. Then,the solid thus obtained was filtered off under reduced pressure. Theresulting solid was dried under reduced pressure to give 9.7 g of2-[2-(methylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (Yield: 52.8%)and was used for the next step without any further processing. ¹H-NMR(DMSO-d6): δ (ppm) 2.16 (s, 3H), 2.84 (t, 2H), 4.29 (t, 2H), 7.87 (s,4H). LC-MS: m/z=238.4 (M+H).

2-[2-(methylsulfonyl)ethoxy]-1H-isoindole-1,3(2H)-dione (I-5)

m-CPBA (11 g) was added portion wise to a stirred solution of2-[2-(methylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (9.6 g) indichloromethane (100 ml) at room temperature and was stirred at roomtemperature for 6 hours. The crude was concentrated under reducedpressure and the resulting residue was suspended in saturated NaHCO₃solution (100 ml) and stirred for 30 minutes. The resulting solid wasfiltered off under reduced pressure and washed with water (50 ml) andwas dried under reduced pressure to give 9.0 g of2-[2-(methylsulfonyl)ethoxy]-1H-isoindole-1,3(2H)-dione (Yield: 82.6%)and was used for the next step without any further processing. ¹H-NMR(DMSO-d6): δ (ppm) 3.15 (s, 3H), 3.66 (t, 2H), 4.54 (t, 2H), 7.88 (s,4H). LC-MS: m/z=270.3 (M+H).

1-(aminooxy)-2-(methylsulfonyl)ethane hydrochloride (I-6)

85% methyl hydrazine (2.0 g) was added drop wise to a stirred suspensionof 2-[2-(methylsulfonyl)ethoxy]-1H-isoindole-1,3(2H)-dione (9.0 g) indichloromethane (100 ml) at room temperature and was stirred for 6hours. Then the reaction mixture was filtered off under reduced pressureto remove insoluble by-product. The resulting filtrate was concentratedunder reduced pressure at lower temperature. The residue was suspendedin 1N HCl (100 ml) and extracted by ethyl acetate (3×250 ml). Theresulting aqueous solution containing the desired product wasconcentrated under reduced pressure to give white solid which wasfurther triturated with diethyl ether and dried under reduced pressureto give 4.0 g of 1-(aminooxy)-2-(methylsulfonyl)ethane hydrochloride(Yield: 68.3%). ¹H-NMR (DMSO-d6): δ (ppm) 3.04 (s, 3H), 3.60 (t, 2H),4.38 (t, 2H), 10.09 (s broad, 2H). LC-MS: m/z=270.3 (M+H).

N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide (I-7)

2N NaOH solution (4.28 ml) was added drop wise to a stirred solution of1-(aminooxy)-2-(methylsulfonyl)ethane hydrochloride (1.5 g) ands-methylisothiosemicarbazide hydroiodide (1.99 g) in water (4.5 ml) atroom temperature and was stirred for 48 hours. Then, the reactionmixture was concentrated under reduced pressure and the residue wasazeotroped with methanol (5 ml). The resulting material was suspended inethanol (10 ml) and insoluble inorganic salts were removed byfiltration. The resulting filtrate which containN′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide was directly usedfor the next step without any further processing.

2-(2-chlorobenzylidene)-N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide(Compound 3)

2-chlorobenzaldehyde (1.32 g) was added drop wise to the filtratecontaining N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide at roomtemperature. The mixture was stirred at room temperature for 2 hours.The reaction mixture was concentrated under reduced pressure and theresidue thus obtained was further purified by Prep HPLC using 0.1%NH₃/water/MeCN to give 20 mg of2-(2-chlorobenzylidene)-N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide(Yield: 0.7% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 3.03 (s, 3H), 3.45(m, 2H), 4.12 (m, 2H), 6.11 (s broad, 2H), 7.40 (m, 2H), 7.44 (m, 1H),8.15 (m, 1H), 8.26 (s broad, 1H), 10.48 (s, 1H). LC-MS: m/z=318.83(M+H).

Compound 4:2-(2-chlorobenzylidene)-N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide2-[3-(methylsulfanyl)propoxy]-1H-isoindole-1,3(2H)-dione (I-8)

Diisopropyl azodicarboxylate (77.92 ml) was added drop wise to a stirredsolution of N-Hydroxyphthalimide (36.8 g), 3-(methylsulfanyl)-1-propanol(30 g) and triphenylphosphine (37.1 g) in anhydrous THF (600 ml) undernitrogen atmosphere at 0° C. The reaction mixture was stirred at 0° C.for 30 minutes and then it was allowed to warm to room temperature andwas stirred for 18 hours. Then, the reaction mixture was concentratedunder reduced pressure to get a crude material which was purified bycolumn chromatography using silica gel. The desired product eluted at 4%ethyl acetate in hexane. Evaporation of pure product fractions gave 30 gof 2-[3-(methylsulfanyl)propoxy]-1H-isoindole-1,3(2H)-dione (Yield:42.2%). ¹H-NMR (DMSO-d6): δ (ppm) 1.94 (q, 2H), 2.07 (s, 3H), 2.67 (t,2H), 4.23 (t, 2H), 7.87 (s, 4H). LC-MS: m/z=252.4 (M+H).

2-[3-(methylsulfonyl)propoxy]-1H-isoindole-1,3(2H)-dione (I-9)

m-CPBA (61.89 g) was added portion wise to a stirred solution of2-[3-(methylsulfanyl)propoxy]-1H-isoindole-1,3(2H)-dione (30.0 g) indichloromethane (550 ml) at room temperature. The mixture was stirred atthe room temperature for 5 hours. Then, the reaction mixtures wasconcentrated under reduced pressure to get a crude material which wassuspended in saturated NaHCO₃ solution (250 ml) and stirred well for 30minutes. The resulting solid was filtered off under reduced pressure andwashed with water (100 ml). The solid was dried under reduced pressureto give 22 g of 2-[3-(methylsulfonyl)propoxy]-1H-isoindole-1,3(2H)-dione(yield: 65%). ¹H-NMR (CDCl3): δ (ppm) 2.32 (m, 2H), 3.00 (s, 3H), 3.50(t, 2H), 4.39 (t, 2H), 7.83 (m, 4H). LC-MS: m/z=283.9 (M+H).

1-(aminooxy)-3-(methylsulfonyl)propane hydrochloride (I-10)

85% methyl hydrazine (4.2 g) was added drop wise to a stirred suspensionof 2-[3-(methylsulfonyl)propoxy]-1H-isoindole-1,3(2H)-dione (20 g) indichloromethane (300 ml) at room temperature and was stirred for 6hours. Then, the solution was filtered off under reduced pressure toremove the insoluble by-product. The resulting filtrate was concentratedunder reduced pressure at low temperature. The residue was suspended in1N HCl (200 ml) and extracted by ethyl acetate (3×500 ml) to removeundesired impurities. The resulting aqueous solution was concentratedunder reduced pressure to give a white solid which was furthertriturated with diethyl ether and dried under reduced pressure to give8.0 g of 1-(aminooxy)-3-(methylsulfonyl)propane hydrochloride (Yield:59.8%). ¹H-NMR (DMSO-d6): δ (ppm) 2.04 (m, 2H), 3.02 (s, 3H), 3.19 (t,2H), 4.12 (t, 2H), 11.06 (s broad, 3H).

N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide (I-11)

2N NaOH solution (5.28 ml) was added drop wise to a stirred solution of1-(aminooxy)-3-(methylsulfonyl)propane hydrochloride (2.0 g) ands-methylisothiosemicarbazide hydroiodide (2.46 g) in water (6.0 ml) atroom temperature. The reaction mixture was stirred at the roomtemperature for 24 hours. Formation ofN-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide was confirmed byLCMS analysis. Then, the mixture was concentrated under reduced pressureand the residue was azeotroped with methanol (15 ml). The resultingmaterial was suspended in ethanol (15 ml) and the insoluble inorganicsalts were removed by filtration. The filtrate was directly used for thenext step without any further processing. LC-MS: m/z=210.8 (M+H).

2-(2-chlorobenzylidene)-N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide(compound 4)

2-chlorobenzaldehyde (1.62 g) was added drop wise to the filtratecontaining N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide atroom temperature. The resulting reaction mixture was stirred at the sametemperature for 2 hours. The crude was concentrated under reducedpressure and the residue thus obtained was further purified by Prep HPLCusing 0.1% NH₃/water/MeCN. After purification, the material was stirredin saturated NaHCO₃ solution and the resulting solid was filtered offunder reduced pressure and washed with water and dried to give 0.14 g ofpure2-(2-chlorobenzylidene)-N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide(Yield: 4% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 2.01 (m, 2H), 2.98(s, 3H), 3.24 (t, 2H), 3.82 (t, 2H), 5.90 (s, 2H), 7.31 (m, 2H), 7.43(d, 1H), 8.13 (m, 2H), 10.48 (s, 1H). LC-MS: m/z=333.5 (M+H).

Compound 5: 2-(2-chlorobenzylidene)-N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide (I-12)

2N NaOH solution (6.8 ml) was added drop wise to a stirred solution ofO-Allylhydroxylamine hydrochloride (1.5 g) ands-methylisothiosemicarbazide hydroiodide (3.22 g) in water (4.2 ml) atroom temperature. The reaction mixture was stirred at room temperaturefor 48 hours. Formation of intermediate I-12N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide was confirmed by LCMSanalysis. Then, the mixture was concentrated under reduced pressure andthe residue was azeotroped with methanol (5 ml). The resulting materialwas suspended in ethanol (10 ml) and the insoluble inorganic salts wereremoved by filtration. The filtrate was directly used for the next stepwithout any further processing. LC-MS: m/z=130.6 (M+H).

2-(2-chlorobenzylidene)-N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide(Compound 5)

2-chlorobenzaldehyde (1.9 g) was added drop wise to the filtratecontaining N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide at roomtemperature and was stirred for 2 hours. The reaction mixture wasconcentrated under reduced pressure and the residue thus obtained wasfurther purified by Prep HPLC using 0.1% HCOOH/water/MeCN to give 0.25 gof2-(2-chlorobenzylidene)-N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide(Yield: 6.1% for 2 steps. ¹H-NMR (DMSO-d6): δ (ppm) 3.17 (s, 1H), 4.23(m, 2H), 5.82 (s broad, 2H), 5.98 (m, 1H), 7.37 (m, 2H), 8.15 (m, 3H).LC-MS: m/z=252.8 (M+H).

Compound 6: 2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy)hydrazinecarboximidamide 2-(2-hydroxyethoxy)-1H-isoindole-1,3(2H)-dione (I-13)

2-Bromotehanol (13.26 ml) was added drop wise to a stirred solution ofN-Hydroxyphthalimide (10.0 g) and Sodium acetate (25.14 g) in DMF (50ml) at room temperature. The resulting reaction mixture was stirred at80° C. for 1.5 hours. The reaction mixture was allowed to cool to roomtemperature and was dumped in 500 ml of cold water and the product wasextracted by ethyl acetate (2×400 ml). The resulting organic layer werecombined and distilled under vacuum. The residue was stirred in coldwater and the resulting solid was filtered off under vacuum. The solidwas dried under reduced pressure to give 6.0 g of2-(2-hydroxyethoxy)-1H-isoindole-1,3(2H)-dione (Yield: 47.3%) which wereused for the next step without any further processing. ¹H-NMR (DMSO-d6):δ (ppm) 3.70 (q, 2H), 4.18 (t, 2H), 4.83 (t, 1H), 7.87 (s, 4H). LC-MS:m/z=208.34 (M+H).

2-(aminooxy)ethanol hydrochloride (I-14)

85% methyl hydrazine (1.25 g) was added drop wise to a stirredsuspension of 2-(2-hydroxyethoxy)-1H-isoindole-1,3(2H)-dione (6.0 g) indichloromethane (25 ml) at room temperature and was stirred for 2 hours.Then, the reaction mixture was filtered off under reduced pressure toremove insoluble by-product. The filtrate was concentrated under reducedpressure at lower temperature. The residue was suspended in 2N HCl inEthylacetate (20 ml) and concentrated under reduced pressure at lowertemperature. The resulting solid was triturated with Dichloromethane(2×15 ml) and dried under reduced pressure to give 2.8 g of2-(aminooxy)ethanol hydrochloride (Yield: 85.5% as mono hydrochloridesalt). ¹H-NMR (DMSO-d6): δ (ppm) 3.61 (m, 2H), 4.04 (t, 2H), 4.73 (m,1H), 11.02 (s broad, 2H).

N′-(2-hydroxyethoxy)hydrazinecarboximidamide (I-15)

2N NaOH solution (10.6 ml) was added drop wise to a stirred solution of2-(aminooxy)ethanol hydrochloride salt (2.4 g) and s-methylisothiosemicarbazide hydroiodide (4.98 g) in water (8.4 ml) at roomtemperature and was stirred for 24 hours. Formation ofN′-(2-hydroxyethoxy)hydrazine carboximidamide was confirmed by LCMSanalysis. The mixtures was concentrated under reduced pressure and theresulting residue was azeotroped with methanol (15 ml). The resultingmaterial was suspended in ethanol (10 ml) and the insoluble inorganicsalts were removed by filtration. The filtrate containingN′-(2-hydroxyethoxy)hydrazinecarboximidamide was directly used for thenext step without any further processing. LC-MS: m/z=134.6 (M+H)

2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy)hydrazinecarboximidamide(Compound 6)

2-chlorobenzaldehyde (3.28 g) was added drop wise to the filtratecontaining N′-(2-hydroxyethoxy)hydrazinecarboximidamide at roomtemperature and was stirred for 2 hours. The reaction mixture wasconcentrated under reduced pressure and the residue thus obtained wasfurther purified by Prep HPLC using 0.1% NH₃/water/MeCN to give 0.24 gof 2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy)hydrazinecarboximidamide(Yield: 4.4% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 3.68 (m, 2H), 3.97(m, 2H), 5.82 (s broad, 2H), 5.07 (m, 1H), 7.50 (m, 2H), 7.55 (m, 1H),8.34 (m, 1H) 8.47 (s, 1H), 8.67 (s, 1H), 11.78 (m, 1H), 12.09 (m, 1H).LC-MS: m/z=256.73 (M+H).

Compound 7: 2-(2-chlorobenzylidene)-N′-(2-chloroethoxy)hydrazinecarboximidamide hydrochloride

SoCl₂ (0.26 ml) was added drop wise to a stirred solution of2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy)hydrazine carboximidamide(0.22 g) in Dichloromethane (10 ml) at 0° C. The reaction mixture wasstirred at the room temperature for 24 hours. Then, the reactionmixtures was concentrated under reduced pressure. The resulting residuewas triturated with n-pentane (2×5 ml) and dried under reduced pressureto give 0.26 g of2-(2-chlorobenzylidene)-N′-(2-chloroethoxy)hydrazinecarboximidamidehydrochloride (Yield: 99.5%). LC-MS: m/z=274.8 (M+H).

Compound 8:2-(2-chlorobenzylidene)-N′-[2-(pyrrolidin-1-yl)ethoxy]hydrazinecarboximidamide

Pyrrolidine (0.23 g) was added to a stirred solution of2-(2-chlorobenzylidene)-N′-(2-chloroethoxy)hydrazine carboximidamidehydrochloride (0.27 g), Triethylamine (0.35 g) and Sodium iodide (0.04g) in THF (10 ml) at room temperature. The resulting mixture was stirredat 50° C. for 24 hours. Then, the reaction mixtures was allowed to coolto room temperature and the crude was dumped in 50 ml of cold water. Theproduct was extracted by ethyl acetate (2×50 ml). Then, organic layerwere combined and distilled under vacuum, the residue thus obtained wasfurther purified by Prep HPLC using 0.1% NH₃/water/MeCN to give 14 mg of2-(2-chlorobenzylidene)-N′-[2-(pyrrolidin-1-yl)ethoxy]hydrazinecarboximidamide(Yield: 5.3%).). ¹H-NMR (MeOD): δ (ppm) 1.91 (m, 4H), 2.75 (m, 4H), 2.88(t, 2H), 3.97 (t, 2H), 7.32 (m, 2H), 7.41 (m, 1H), 8.07 (m, 1H), 8.32(s, 1H). LC-MS: m/z=310.33 (M+H).

Compound 10: 2-(2-chlorobenzylidene)-N′-ethoxyhydrazinecarboximidamideN′-(2-ethoxy)hydrazinecarboximidamide (I-16)

1N NaOH solution (5.12 ml) was added drop wise to a stirred solution ofethoxyamine hydrochloride salt (0.5 g) and s-methyl isothiosemicarbazidehydroiodide (1.19 g) in water (5.0 ml) at room temperature and wasstirred for 48 hours. Formation of N′-(2-ethoxy)hydrazinecarboximidamidewas confirmed by LCMS analysis. The mixtures was concentrated underreduced pressure and the resulting residue was dissolved in ethanol (15ml). The insoluble solids were removed by filtration. The filtrate wasconcentrated and N′-(2-ethoxy)hydrazinecarboximidamide was directly usedfor the next step without any further processing. LC-MS: m/z=118.8(M+H).

2-(2-chlorobenzylidene)-N′-ethoxyhydrazinecarboximidamide (compound 10)

2-chlorobenzaldehyde (0.717 g) was added dropwise toN′-(2-ethoxy)hydrazinecarboximidamide in solution in ethanol (10 ml) andsodium acetate (0.42 g) at room temperature and was stirred for 2 hoursat 90° C. The reaction mixture was concentrated under reduced pressureand the residue thus obtained was further purified by chromatography togive 21.4 mg of2-(2-chlorobenzylidene)-N′-(2-ethoxy)hydrazinecarboximidamide (Yield:1.7% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 1.18 (t, 3H), 3.77 (q, 2H),5.77 (s broad, 2H), 7.31 (m, 2H), 7.43 (m, 1H), 8.11 (m, 1H), 8.15 (s,1H), 10.45 (s broad, 1H). LC-MS: m/z=240.9 (M+H).

Compound 11:2-(2,6-dichlorobenzylidene)-N-ethoxyhydrazinecarboximidamide

2,6-dichlorobenzaldehyde (0.896 g) was added dropwise to 1 equivalent ofN′-(2-ethoxy)hydrazinecarboximidamide (I-16) in solution in ethanol (10ml) and sodium acetate (0.42 g) at room temperature and was stirred for2 hours at 90° C. The reaction mixture was concentrated under reducedpressure and the residue thus obtained was further purified bychromatography to give 57 mg of2-(2,6-dichlorobenzylidene)-N′-(2-ethoxy)hydrazinecarboximidamide(Yield: 4.1% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 1.77 (t, 3H), 3.78(q, 2H), 5.48 (s broad, 2H), 7.33 (t, 1H), 7.52 (m, 2H), 8.04 (s, 1H),8.16 (m, 1H). LC-MS: m/z=277.1 (M+H).

Compound 12: 2-(2-chlorobenzylidene)-N-propoxyhydrazinecarboximidamideN′-propoxyhydrazinecarboximidamide (I-17)

2N NaOH solution (1.23 ml) was added dropwise to a stirred solution ofO-propylhydroxylamine hydrochloride salt (0.28 g) and s-methylisothiosemicarbazide hydroiodide (0.58 g) in water (2.0 ml) at roomtemperature and was stirred for 24 hours. Formation ofN′-(propoxy)hydrazinecarboximidamide was confirmed by LCMS analysis. Themixtures was concentrated under reduced pressure and the resultingresidue was dissolved in ethanol (15 ml). The insoluble solids wereremoved by filtration. The filtrate was concentrated andN′-(propoxy)hydrazinecarboximidamide was directly used for the next stepwithout any further processing. LC-MS: m/z=132.9 (M+H)

2-(2-chlorobenzylidene)-N-propoxyhydrazinecarboximidamide (compound 12)

2-chlorobenzaldehyde (0.35 g) was added dropwise toN′-(2-propoxy)hydrazinecarboximidamide in solution in ethanol (10 ml)and was stirred for 2 at room temperature. The reaction mixture wasconcentrated under reduced pressure and the residue thus obtained wasfurther purified by chromatography to give 25 mg of2-(2-chlorobenzylidene)-N′-(2-propoxy)hydrazinecarboximidamide (Yield:3.9% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 0.88 (t, 3H), 1.58 (m, 2H),3.66 (t, 2H), 5.75 (s broad, 1H), 7.29 (m, 2H), 7.41 (m, 1H), 8.10 (m,2H), 10.45 (s broad, 2H). LC-MS: m/z=255.1 (M+H).

Compound 13: 2-(2-chlorobenzylidene)-N-(2-ethoxyethoxy)hydrazinecarboximidamide2-(2-ethoxyethoxy)-1,3-dimethylidene-2,3-dihydro-1H-isoindole (I-18)

The N-hydroxypthalimide (4.0 g) and 1-bromo-2-ethoxyethane (11.25 g)were dissolved in DMF (40.0 ml) and CH₃COONa (10.0 g) was added to thesolution at room temperature. The reaction mixture was allowed to stirat 70° C. for 12 hours. The reaction mixture was allowed to cool to roomtemperature and was and was poured in water and then extracted two timesby ethyl acetate. The organic layer was concentrated under reducepressure and was purified by column chromatography using silica gel. Thedesired product was eluted with 0-30% ethyl acetate in hexane.Evaporation of pure product fractions gave 4.8 g of2-(2-ethoxyethoxy)-1,3-dimethylidene-2,3-dihydro-1H-isoindole (I-18)(Yield: 83.3%). ¹H-NMR (DMSO-d6): δ (ppm) 0.98 (t, 3H), 3.39 (q, 2H),3.73 (t, 2H), 4.27 (t, 2H), 7.87 (s, 4H). LC-MS: m/z=236.2 (M+H).

1-(aminooxy)-2-ethoxyethane hydrochloride (I-19)

Hydrazine hydrate (1.32 g) was added dropwise to a stirred solution of2-(2-ethoxyethoxy)-1,3-dimethylidene-2,3-dihydro-1H-isoindole (4.8 g) inmethanol (10 ml) at room temperature and was stirred for 30 minutes.Then, the reaction mixture was filtered off under reduced pressure toremove insoluble by-product. The filtrate was concentrated under reducedpressure at lower temperature and triturated ether and insoluble wasremoved by filtration. Then, to the filtrate, 4N HCl in dioaxane (10.2ml) was added dropwise and the precipitated salt was collected byfiltration and was dried to 2.0 g of 1-(aminooxy)-2-ethoxyethanehydrochloride (Yield: 69.4% as mono hydrochloride salt). ¹H-NMR(DMSO-d6): δ (ppm) 1.11 (t, 3H), 3.44 (q, 2H), 3.59 (m, 2H), 4.14 (m,2H), 11.02 (s broad, 2H). LC-MS: m/z=106.1 (M+H).

N′-(2-ethoxyethoxy)hydrazinecarboximidamide (I-20)

1N NaOH solution (4.23 ml) was added dropwise to a stirred solution of1-(aminooxy)-2-ethoxyethane hydrochloride salt (0.6 g) and s-methylisothiosemicarbazide hydroiodide (0.99 g) in water (2.1 ml) at roomtemperature and was stirred for 48 hours. Formation ofN′-(2-ethoxyethoxy)hydrazinecarboximidamide was confirmed by LCMSanalysis. The mixtures was concentrated under reduced pressure and theresulting residue was dissolved in ethanol (10 ml). The insoluble solidswere removed by filtration. The filtrate was concentrated andN′-(2-ethoxyethoxy)hydrazinecarboximidamide was directly used for thenext step without any further processing. LC-MS: m/z=163.0 (M+H).

2-(2-chlorobenzylidene)-N-(2-ethoxyethoxy)hydrazinecarboximidamide(compound 13)

2-chlorobenzaldehyde (0.59 g) was added dropwise to N′-(2-ethoxyethoxy)hydrazinecarboximidamide in solution in ethanol (5 ml) and was stirredfor 2 at room temperature. The reaction mixture was concentrated underreduced pressure and the residue thus obtained was further purified bychromatography to give 19 mg of2-(2-chlorobenzylidene)-N′-(2-propoxy)hydrazinecarboximidamide (Yield:1.8% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 1.24 (t, 3H), 3.48 (q, 2H),3.56 (m, 2H), 3.83 (m, 2H), 5.80 (s broad, 1H), 7.43 (m, 1H), 8.12 (m,1H), 8.17 (s, 1H), 10.50 (s broad, 2H). LC-MS: m/z=285.0 (M+H).

Compound 14:2-(2-chlorobenzylidene)-N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide2-[(3-methylbut-2-en-1-yl)oxy]-1H-isoindole-1,3(2H)-dione (I-21)

Triethylamine (12.13 g) was added dropwise to a stirred solution ofN-Hydroxyphthalimide (9.85 g) and 1-bromo-3-methyl butene (9.0 g) in DMF(30 ml) at room temperature. The reaction mixture was stirred at 70° C.for 2 hours. The reaction mixture was allowed to cool to roomtemperature. The mixture was concentrated under reduced pressure and theresidue thus obtained was suspended in cold water. The resultingsuspension was stirred well for some time and the solid was filtered offunder reduced pressure. The solid was further washed with demineralizedwater (200 ml) and hexane (100 ml). The resulting solid was dried underreduced pressure to get a crude material which was purified by columnchromatography using silica gel to give 9.0 g of-[(3-methylbut-2-en-1-yl)oxy]-1H-isoindole-1,3(2H)-dione (Yield: 64.5%).¹H-NMR (DMSO-d6): δ (ppm) 1.70 (d, 6H), 4.63 (m, 2H), 5.45 (m, 1H), 7.87(s, 4H). LC-MS: m/z=232.1 (M+H).

1-(aminooxy)-3-methylbut-2-ene hydrochloride (I-22)

Hydrazine hydrate (2.52 g) was added dropwise to a stirred solution of2-[(3-methylbut-2-en-1-yl)oxy]-1H-isoindole-1,3(2H)-dione (9.0 g) inmethanol (120 ml) at room temperature. The reaction mixture was stirredat the same temperature for 30 min. The reaction mixture was filteredoff to remove the insoluble by-product and the resulting filtrate wasconcentrated under reduced pressure to get a crude material which waspurified by column chromatography using silica gel. The crude wastriturated with ether and insoluble mass was removed by filtration. Thefiltrate was treated with 4 M HCl in dioxane (19 ml) dropwise and theprecipitate was filtered, collected and dried under vacuum to give 2.9 gof 1-(aminooxy)-3-methylbut-2-ene hydrochloride (Yield: 73.6%). ¹H-NMR(DMSO-d6): δ (ppm) 1.70 (s, 3H), 1.75 (s, 3H), 1.65 (m, 1H), 4.50 (d,2H), 5.30 (t, 1H), 10.89 (s, 3H).

N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide (I-23)

1N NaOH solution (3.63 ml) was added dropwise to a stirred solution of1-(aminooxy)-3-methylbut-2-ene hydrochloride (0.5 g) ands-methylisothiosemicarbazide hydro-iodide (0.85 g) in water (3 ml) atroom temperature and was stirred for 48 hours. Then, the reactionmixtures was concentrated under reduced pressure. The resulting residuewas suspended in ethanol (15 ml) and insoluble inorganic salts wereremoved by filtration. The filtrate was concentrated and directly usedfor the next step without any further processing.N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide was confirmed byLCMS analysis. LC-MS: m/z=159.15 (M+H).

2-(2-chlorobenzylidene)-N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide(compound 14)

2-chlorobenzaldehyde (0.5 g) was added dropwise toN′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide in solution inethanol (3 ml) at room temperature and was stirred for 2 hours at 90° C.The reaction mixture was concentrated under reduced pressure and theresidue thus obtained was further purified by chromatography to give 139mg of2-(2-chlorobenzylidene)-N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide(Yield: 13.5% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 1.64 (s, 3H), 1.71(s, 3H), 3.17 (s, 1H), 4.25 (d, 2H), 5.39 (t, 1H), 5.75 (s broad, 2H),7.32 (m, 1H), 7.43 (m, 1H), 8.10 (m, 1H), 8.15 (m, 1H), 8.17 (s broad,1H). LC-MS: m/z=281.2 (M+H).

Compound 15: 2-(2-chlorobenzylidene)-N-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide 2-bromoethyl ethyl sulphide (I-24)

PBr₃ (10 ml) was added dropwise to 2-(ethylsulfanyl)ethanol in solutionin dichloromethane (100 ml) at 0° C. and was stirred for 2 hours. Thenthe reaction mixture was warmed to room temperature and stirred for 16hours. The reaction mixture was cooled at 0° C. and 10 ml of water wasadded. Then reaction mixture was neutralized with saturated Na₂CO₃solution (˜up to Ph 7) and extracted with dichloromethane (3×250 ml).The organic layers were separated, combined and dried (Na₂SO₄) andconcentrated to afford 13.0 g of 2-bromoethyl ethyl sulphide (yield:72.7%). ¹H-NMR (CDCl₃): δ (ppm) 1.30 (t, 3H), 2.62 (q, 2H), 2.97 (m,2H), 3.50 (m, 2H).

2-[2-(ethylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (I-25)

The N-hydroxypthalimide (3.9 g) and 2-bromoethyl ethyl sulphide (12.1 g)were dissolved in DMF (40.0 ml) and CH₃COONa (9.7 g) was addedportionwise to the solution at room temperature. The reaction mixturewas allowed to stir at 70° C. for 2 hours. The reaction mixture wasallowed to cool to room temperature and was and was poured in cold waterand then extracted two times by ethyl acetate. The organic layer wasconcentrated under reduce pressure and was purified by columnchromatography using silica gel. To give 6.0 g of2-[2-(ethylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (I-25) (Yield:98%). ¹H-NMR (CDCl₃): δ (ppm) 1.29 (t, 3H), 2.63 (q, 2H), 2.94 (t, 2H),4.36 (t, 2H), 7.77 (m, 2H), 7.86 (m, 2H).

1-(aminooxy)-2-(ethylsulfanyl)ethane hydrochloride (I-26)

Hydrazine hydrate (0.25 g) was added dropwise to a stirred solution of2-[2-(ethylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (1.0 g) inmethanol (10 ml) at room temperature. The reaction mixture was stirredat the same temperature for 30 min. The reaction mixture was filteredoff to remove the insoluble by-product and the resulting filtrate wasconcentrated under reduced pressure then dissolved in DCM and insolubleremoved by filtration. The filtrate was concentrated under reducedpressure then, the crude was triturated with ether and insoluble masswas removed by filtration. The filtrate was treated with 4 M HCl indioxane (2 ml) dropwise. Then the solvent was removed by evaporation andthe the residue was triturated with diethyl ether to provide 454 mg1-(aminooxy)-2-(ethylsulfanyl)ethane hydrochloride (Yield: 72.5%).¹H-NMR (DMSO-d6): δ (ppm) 1.18 (s, 3H), 2.53 (m, 2H), 2.79 (t, 2H), 4.16(t, 2H), 11.14 (s broad, 3H).

N′-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide (I-27)

1N NaOH solution (2.88 ml) was added dropwise to a stirred solution of1-(aminooxy)-2-(ethylsulfanyl)ethane hydrochloride (0.5 g) and s-methylisothiosemicarbazide hydroiodide (0.7 g) in water (5 ml) at roomtemperature and was stirred for 48 hours. The mixtures was concentratedunder reduced pressure and the resulting residue was dissolved inethanol (15 ml). The insoluble solids were removed by filtration. Thefiltrate was concentrated andN′-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide was directly usedfor the next step without any further processing.

2-(2-chlorobenzylidene)-N-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide(compound 15)

2-chlorobenzaldehyde (0.4 g) was added dropwise toN′-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide in solution inethanol (5 ml) and was stirred for 2 at room temperature. The reactionmixture was concentrated under reduced pressure and the residue thusobtained was further purified by chromatography to give 15 mg of2-(2-chlorobenzylidene)-N-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide(Yield: 1.5% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 1.90 (t, 3H), 2.54(q, 2H), 2.75 (t, 2H), 3.85 (t, 2H), 5.84 (s broad, 2H), 7.30 (m, 2H),7.44 (m, 1H), 8.12 (m, 1H), 8.16 (s, 1H), 10.50 (s broad, 1H). LC-MS:m/z=301.9 (M+H).

Compound 16:2-[(3-chloropyridin-4-yl)methylidene]-N-ethoxyhydrazinecarboximidamide

3-chloroisonicotinaldehyde (0.72 g) was added dropwise to 1 equivalentof N′-(2-ethoxy)hydrazinecarboximidamide (I-16) in solution in ethanol(5 ml) and sodium acetate (0.42 g) at room temperature and was stirredfor 2 hours at 80° C. The reaction mixture was concentrated underreduced pressure and the residue thus obtained was further purified bychromatography to give 184 mg of2-[(3-chloropyridin-4-yl)methylidene]-N-ethoxyhydrazinecarboximidamide(Yield: 15% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 1.19 (t, 3H), 3.79(q, 2H), 5.96 (s broad, 2H), 8.05 (s, 1H), 8.11 (d, 1H), 8.41 (s, 1H),10.89 (s broad, 1H). LC-MS: m/z=242.0 (M+H).

Compound 17:2-(2-chloro-6-fluorobenzylidene)-N-ethoxyhydrazinecarboximidamide

2-chloro-6-flurobenzaldehyde (0.81 g) was added dropwise to 1 equivalentof N′-(2-ethoxy)hydrazinecarboximidamide (I-16) in solution in ethanol(5 ml) and sodium acetate (0.42 g) at room temperature and was stirredfor 2 hours at 80° C. The reaction mixture was concentrated underreduced pressure and the residue thus obtained was further purified bychromatography to give 215 mg of2-(2-chloro-6-fluorobenzylidene)-N-ethoxyhydrazinecarboximidamide(Yield: 17.2% for 2 steps). ¹H-NMR (DMSO-d6): δ (ppm) 1.17 (m, 3H), 3.78(q, 2H), 5.48 (s broad, 2H), 7.30 (m, 3H), 8.01 (s, 1H), 10.54 (s broad,1H). LC-MS: m/z=258.9 (M+H).

Compound 18: N′-butoxy-2-(2-chlorobenzylidene)hydrazinecarboximidamide

Compound 18 is prepared following the same procedure than compound 12from 2-chlorobenzaldehyde and N′-(2-butoxy)hydrazinecarboximidamide.

Compound 19:2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamide

Compound 19 is prepared following the same procedure than compound 17from 2-chloro-6-flurobenzaldehyde and N′-(2-propoxy)hydrazinecarboximidamide (I-17) to give2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamideLC-MS: m/z=273.0 (M+H).

Compound 20:2-(2-chloro-6-fluorobenzylidene)-N′-butoxyhydrazinecarboximidamide

Compound 20 is prepared following the same procedure than compound 17from 2-chloro-6-flurobenzaldehyde andN′-(2-butoxy)hydrazinecarboximidamide.

Compound 21:2-(2,6-dichlorobenzylidene)-N-propoxyhydrazinecarboximidamide

Compound 21 is prepared following the same procedure than compound 11from 2,6-dichlorobenzaldehyde and N′-(2-propoxy)hydrazinecarboximidamide(I-17) to give2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamideLC-MS: m/z=290.9 (M+H).

Compound 22:2-(2,6-dichlorobenzylidene)-N-butoxyhydrazinecarboximidamide

Compound 22 is prepared following the same procedure than compound 17from 2,6-dichlorobenzaldehyde and N′-(2-butoxy)hydrazinecarboximidamide.

Compound 23:2-[(3-chloropyridin-4-yl)methylidene]-N-propoxyhydrazinecarboximidamide

Compound 23 is prepared following the same procedure than compound 16from 3-chloroisonicotinaldehyde andN′-(2-propoxy)hydrazinecarboximidamide (I-17) to give2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamideLC-MS: m/z=255.9 (M+H).

Selected compounds according to the invention are set forth in Tablebelow:

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

In some of the experiments below, the salt of these compounds may beused.

1.2—Mammalian Cell Culture, Constructs and Transfection

HeLa Cells were cultured in Eagle's Minimum Essential Medium (EMEM)supplemented with Glutamine, Sodium Pyruvate, Non-Essential Amino Acids,Penicillin and Streptomycin (Lonza) containing 10% Foetal Bovine Serum(FBS) (Biowest). 293T cells were cultured in Dubelcco's Modified Eagle'sMedia (DMEM) supplemented with penicillin, streptomycin, glutamine(Lonza) and 10% of fetal bovine serum (FBS) (Biowest).

Min6 cells were cultured in DMEM supplemented with penicillin,streptomycin, glutamine, sodium pyruvate, 50 μM β-Mercaptoethanol and15% Foetal Bovine Serum (FBS) (Biowest).

INS1 cells were cultured in RPMI supplemented with penicillin,streptomycin, glutamine, sodium pyruvate (Lonza), 50 μMβ-Mercaptoethanol and 10% of fetal bovine serum (FBS) (Biowest).

Each cell line was maintained at 37° C. in 5% CO₂ atmosphere.

Human open reading frame (ORF) sequences for PLP1, DM20 and Insulin wereobtained from Life Technologies (Invitrogen) (IOH41689, IOH5252 andIOH7334 respectively). Construct cloning into the expression plasmidpDEST26 (Invitrogen) was performed by Gateway® LR Clonase™ II Enzyme Mix(Invitrogen). ORF mutations were carried out using the QuikChangeLightning Site-Directed Mutagenesis Kit (Stratagene) (T181P mutation forPLP1 and DM20 ORFs, Akita (C96Y) for Insulin ORF).

Gene expression into mammalian cells was carried out by nucleofection,using the Amaxa™ 4D-Nucleofector™ System (Lonza) or by transfectionusing Lipofectamine (Life technologies).

1.3—Cytoprotection from ER Stress

This assay is described in Tsaytler et al. (Science 2011). HeLa Cellswere cultured in Eagle's Minimum Essential Medium (EMEM) supplementedwith Glutamine, Sodium Pyruvate, Non-Essential Amino Acids, Penicillinand Streptomycin containing 10% Foetal Bovine Serum (FBS), at 37° C. in5% CO₂ atmosphere. Cells were plated in 96 well plates at a density of17,000 cells/mL the day before the treatment. ER stress was elicited byaddition of 5 μg/mL tunicamycin (Sigma-Aldrich) together with PPP1R15Ainhibitors (0.5-10 μM). Media were changed 6 h later with fresh mediaand the cytoprotection was maintained by the addition of PPP1R15Ainhibitors (0.5-10 μM). Cell viability was assessed by measuring thereduction of WST-8 into formazan using Cell Counting Kit-8 (Sigma)according to the supplier's recommendation, 48 h or 72 h aftertunicamycin treatment. Cytoprotection from ER stress is measured interms of cytoprotective potency effect compared to the referencecompound Guanabenz (Tsaytler et al., Science 2011) after ER stress:

-   -   ‘−’ no cytoprotective effect;    -   ‘+’ lower cytoprotective effect compared to Guanabenz;    -   ‘++’ similar cytoprotective effect compared to Guanabenz;    -   ‘+++’ higher cytoprotective effect compared to Guanabenz.

Table 1 summarizes the results of cytoprotective effect of differentcompounds of the invention, compared to guanabenz, after the stressinduced by a 6 hour exposure of tunicamycin.

1.4—Assessment of Translation Rates in Unstressed Cells

HeLa cells (100,000 cells/ml) were plated in 6-well plates 24 h beforeeach experiment and were either left untreated or treated with compounds(50 μM) for 2.5, 5 and 9 h. Culture medium was replaced bymethionine-free DMEM medium (Invitrogen) 30 min before compoundsaddition. One hour before each time point, 50 μM of Click-iT® AHA(L-azidohomoalanine) (Invitrogen) was added to the culture medium inorder to label newly synthesized proteins. At the end of each timepoint, cells were washed with ice-cold PBS and harvested by Trypsinedissociation (Lonza), then lysed in a 50 mM Tris-HCl buffer containing1% of SDS (Sigma) and protease and phosphatase inhibitors (Sigma).Protein samples were coupled to alkyne biotin (Invitrogen) usingClick-iT® Protein Reaction Buffer Kit (Invitrogen). Samples weredenatured at 70° C. for 10 min, resolved on ECL 4-20% precasted gels (GEHealthcare) and transferred to nitrocellulose membranes (GE Healthcare).Alkyne biotin coupled to Click-iT® AHA incorporated to newly synthesizedproteins was detected using streptavidin-HRP (Gentex). Revelation wasperformed by incubation of ECL Prime (GE Healthcare) and read bychemoluminiscence using Fusion Solo 3S (Vilber Lourmat).

1.5—Assessment of Translation Rates in Stressed Cells

Treatments were performed as for measuring translation in unstressedcells, except that Tunicamycin (5 μg/ml) was added together with thecompounds.

1.6—Functional GPCR Assay for Adrenergic α2A Receptor (CellKey DetectionMethod)

The agonist activity of compounds was evaluated on CHO cellsendogenously expressing human alpha2A receptor and was determined bymeasuring their effects on impedance modulation using the CellKeydetection method. Cells were seeded onto 96-well plate at density of6×10⁴ cells/well in HBSS buffer (Invitrogen)+20 mM HEPES (Invitrogen)with 0.1% BSA and are allowed to equilibrate for 60 min at 28° C. beforethe start of the experiment. Plates were placed onto the system andmeasurements were made at a temperature of 28° C. Solutions were addedsimultaneously to all 96 wells using an integrated fluidics system: HBSS(basal control), reference agonist at 100 nM (stimulated control),reference agonist (EC₅₀ determination) or the test compounds. Impedancemeasurements are monitored for 10 minutes after ligand addition. Thestandard reference agonist is epinephrine, which is tested in eachexperiment at several concentrations to generate aconcentration-response curve from which its EC₅₀ value is calculated.

Dose-response data from test compounds were analysed with Hill softwareusing non-linear regression analysis of the concentration-responsecurves generated with mean replicate values using Hill equation curvefitting. Results are presented table 1, compounds with EC50>33.3 μM areconsidered to have no significant alpha-2 adrenergic activity.

1.7—In Vitro Multiple Sclerosis Disease Model: Interferon-Gamma InjuredRat Oligodendrocytes Co-Cultured with Neurons

Culture of Oligodendrocyte Co-Cultured with Neurons

Neurons/OPC were cultured as previously describes by Yang et al. (2005 JNeurosci Methods; 149(1) pp 50-6) with modifications. Briefly, the fullbrain (without cerebellum) obtained from 17-day old rat embryos (Wistar,Janvier labs) were removed. The full brains were treated for 20 min at37° C. with a trypsin-EDTA (Pan Biotech) solution at a finalconcentration of 0.05% trypsin and 0.02% EDTA. The dissociation wasstopped by addition of Dulbecco's modified Eagle's medium (DMEM) with4.5 g/liter of glucose (Pan Biotech), containing DNAse I grade II (finalconcentration 0.5 mg/ml; Pan Biotech, Batch: h140508) and 10% fetal calfserum (FCS; Invitrogen, Batch: 41Q7218K). Cells were mechanicallydissociated by three forced passages through the tip of a 10-ml pipette.Cells were then centrifuged at 515 g for 10 min at 4° C. The supernatantwas discarded, and the pellet was resuspended in a defined culturemedium consisting of Neurobasal medium (Invitrogen, Batch: 1636133) witha 2% solution of B27 supplement (Invitrogen, Batch: 1660670), 2mmol/liter of L-glutamine (Pan Biotech), 2% of PS solution, and, 1% ofFCS and 10 ng/ml of platelet derived growth factor (PDGF-AA, Batch:H131205). The cells were seeded at a density of 20 000 cells per well in96 well plates precoated with PLL (BD corning, Batch: 6614022) andlaminine (Sigma, Batch: 083M4034V). The plates were maintained at 37° C.into a humidified incubator, in an atmosphere of air (95%)-CO2 (5%).Half of the medium was changed every 2 days with fresh medium. On days18, test compounds were pre-incubated 1 hour before interferon-gamma (70U/ml, 48H, R&D system, Batch: AAL2214081) application.

Test Compounds and Interferon-Gamma Exposure

On day 18 of culture, test compounds (4 concentrations) were solved inculture medium and then pre-incubated with oligodendrocyte co-culturedwith neurons for 1 hour before the interferon-gamma (70 U/ml, 48H)application. One hour after test compounds incubation, interferon-gammawas added at 70 U/ml concentration for 48 H still in presence of testcompounds. Then, cells were fixed by a cold solution of ethanol (95%,Sigma, Batch: SZBD3080V) and acetic acid (5%, Sigma, Batch: SZBD1760V)for 5 min at −20° C. After permeabilization with 0.1% of saponin (Sigma,Batch: BCBJ8417V), cells were incubated for 2 h with Monoclonal Anti-O4antibody produced in mouse (Sigma, batch: SLBF5997V) at dilution of1/1000 in PBS (PAN, Batch: 8410813) containing 1% FCS, 0.1% saponin, for2 h at room temperature. This antibody are revealed with Alexa Fluor 488goat anti-mouse IgG (Invitrogen, batch: 1664729) at the dilution 1/400in PBS containing 1% FCS, 0.1% saponin, for 1 h at room temperature.

Analysis of Total Number of O4 Cells

For each condition, 30 pictures per well were taken using ImageXpress(Molecular Device) with 20× magnification. All images were taken withthe same conditions. Analysis of total number of O4 cells was performedautomatically by using Custom module editor (Molecular Device). Datawere expressed in percentage of control conditions (no intoxication, nointerferon-gamma=100%) in order to express the interferon-gamma injury.All values were expressed as mean+/−SEM (s.e.mean) (n=6 wells percondition).

1.8—In Vitro Parkinson's Disease Model: Rotenone Injured PrimaryMesencephalic Rat Neurons

Culture of Mesencephalic Dopaminergic Neurons

Rat dopaminergic neurons were cultured as described by Schinelli et al.,(1988 J. Neurochem 50 pp 1900-07) and Visanji et al., (2008 FASEB J.22(7) pp 2488-97). Briefly, the midbrains obtained from 15-day old ratembryos (Janvier Labs, France) were dissected under a microscope. Theembryonic midbrains were removed and placed in ice-cold medium ofLeibovitz (L15, Pan Biotech, Batch: 9310614) containing 2% ofPenicillin-Streptomycin (PS, Pan Biotech, Batch: 1451013) and 1% ofbovine serum albumin (BSA, Pan Biotech, Batch: h140603). The ventralportion of the mesencephalic flexure, a region of the developing brainrich in dopaminergic neurons, was used for the cell preparations.

The midbrains were dissociated by trypsinisation for 20 min at 37° C.(Trypsin 0.05% EDTA 0.02%, PanBiotech, Batch: 5890314). The reaction wasstopped by the addition of Dulbecco's modified Eagle's medium (DMEM,PanBiotech, Batch: 1300714) containing DNAase I grade II (0.1 mg/ml,PanBiotech, Batch: H140508) and 10% of foetal calf serum (FCS, Gibco,Batch: 41Q7218K). Cells were then mechanically dissociated by 3 passagesthrough a 10 ml pipette. Cells were then centrifuged at 180×g for 10 minat +4° C. on a layer of BSA (3.5%) in L15 medium. The supernatant wasdiscarded and the cell pellets were re-suspended in a defined culturemedium consisting of Neurobasal (Invitrogen, Batch: 1636133)supplemented with B27 (2%, Invitrogen, Batch: 1660670), L-glutamine (2mM, PanBiotech, Batch: 8150713) and 2% of PS solution and 10 ng/ml ofBrain-derived neurotrophic factor (BDNF, PanBiotech, Batch: H140108) and1 ng/ml of Glial-Derived Neurotrophic Factor (GDNF, Pan Biotech, Batch:H130917). Viable cells were counted in a Neubauer cytometer using thetrypan blue exclusion test. The cells were seeded at a density of 40 000cells/well in 96 well-plates pre-coated with poly-L-lysine (CorningBiocoat, Batch: 6614022) and maintained in a humidified incubator at 37°C. in 5% CO₂/95% air atmosphere. Half of the medium was changed every 2days with fresh medium.

On day 6 of culture, the medium was removed and fresh medium was added,without or with rotenone (Sigma, Batch: 021M2227V) at 10 nM diluted incontrol medium, 3 wells per condition were assessed. Test compounds weresolved in culture medium and then pre-incubated with mesencephalicneurons for 1 hour before the rotenone application.

After 24 hours of intoxication, cells were fixed by a solution of 4%paraformaldehyde (Sigma, batch SLBF7274V) in PBS (Pan Biotech, Batch:4831114), pH=7.3 for 20 min at room temperature. The cells were washedagain twice in PBS, and then were permeabilized and non-specific siteswere blocked with a solution of PBS containing 0.1% of saponin (Sigma,batch: BCBJ8417V) and 1% FCS for 15 min at room temperature. Then, cellswere incubated with Monoclonal Anti-Tyrosine Hydroxylase antibodyproduced in mouse (TH, Sigma, batch: 101M4796) at dilution of 1/10 000in PBS containing 1% FCS, 0.1% saponin, for 2 h at room temperature.This antibody was revealed with Alexa Fluor 488 goat anti-mouse IgG(Molecular Probes, batch: 1531668) at the dilution 1/800 in PBScontaining 1% FCS, 0.1% saponin, for 1 h at room temperature.

Analysis of Total Number of TH Positive Neurons

The immunolabeled cultures were automatically examined with ImageXpress(Molecular device USA). For each condition, 20 automatically fields perwell (representing ˜80% of the total surface of the well) from 3 wellswere analyzed. The total number of TH neurons was automatically analyzedusing Custom module editor (Molecular Devices, USA). Data were expressedin percentage of control conditions (no intoxication, no rotenone=100%)in order to express the rotenone injury. All values were expressed asmean+/−SEM (s.e. mean) of the 1 culture (n=3 wells per condition perculture).

1.9—In Vitro Alzheimer Disease Model: Amyloid-Beta 1-42 Injured PrimaryCortical Rat Neurons.

Culture of Rat Cortical Neurons

Rat cortical neurons were cultured as described by Singer et al., (1999J. Neuroscience 19 pp 2455-63) and Callizot et al., (2013 J. Neurosci.Res. 91 pp 706-16). Pregnant females (Wistar; Janvier Labs) at 15 daysof gestation were killed by cervical dislocation. Fetuses were collectedand immediately placed in ice-cold L15 Leibovitz medium (Pan Biotech,Batch: 9310614) with a 2% penicillin (10,000 U/ml) and streptomycin (10mg/ml) solution (PS; Pan Biotech, Batch: 1451013) and 1% bovine serumalbumin (BSA; Pan Biotech, Batch: h140603). Cortex was treated for 20min at 37° C. with a trypsin-EDTA (Pan Biotech, Batch: 5890314) solutionat a final concentration of 0.05% trypsin and 0.02% EDTA. Thedissociation was stopped by addition of Dulbecco's modified Eagle'smedium (DMEM) with 4.5 g/liter of glucose (Pan Biotech, batch: 1300714),containing DNAse I grade II (final concentration 0.5 mg/ml; Pan Biotech,Batch: h140508) and 10% fetal calf serum (FCS; Invitrogen, Batch:41Q7218K). Cells were mechanically dissociated by three forced passagesthrough the tip of a 10-ml pipette. Cells were then centrifuged at 515 gfor 10 min at 4° C. The supernatant was discarded, and the pellet wasresuspended in a defined culture medium consisting of Neurobasal medium(Invitrogen, Batch: 1636133) with a 2% solution of B27 supplement(Invitrogen, Batch: 1660670), 2 mmol/liter of L-glutamine (Pan Biotech,Batch: 8150713), 2% of PS solution, and 10 ng/ml of brain-derivedneurotrophic factor (BDNF; Pan Biotech, Batch: H140108). Viable cellswere counted in a Neubauer cytometer, using the trypan blue exclusiontest. The cells were seeded at a density of 30,000 per well in 96-wellplates precoated with poly-L-lysine (Corning Biocoat, Batch: 6614022)and were cultured at 37° C. in an air (95%)-CO₂ (5%) incubator. Themedium was changed every 2 days. The cortical neurons were intoxicatedwith A-beta solutions (see below) after 11 days of culture.

Test Compounds and Amyloid-Beta 1-42 Exposure

The Amyloid-beta1-42 preparation was done following the proceduredescribed by Callizot et al., 2013. Briefly, Amyloid-beta 1-42 peptide(Bachem, Batch: 1014012) was dissolved in the defined culture mediummentioned above, devoid of serum, at an initial concentration of 40μmol/liter. This solution was agitated for 3 days at 37° C. in the darkand immediately used after being properly diluted in culture medium tothe concentrations used. Test compounds were solved in culture mediumand then pre-incubated with primary cortical neurons for 1 hour beforethe Amyloid-beta 1-42 application. Amyloid-beta 1-42 preparation wasadded to a final concentration of 20 μM (including to ˜2 μM of toxicoligomers measured by WB) diluted in control medium in presence ofdrugs. After 24 hours of intoxication, cells were fixed by a coldsolution of ethanol (95%, Sigma, Batch: SZBD3080V) and acetic acid (5%,Sigma, Batch: SZBD1760V) for 5 min at −20° C. After permeabilizationwith 0.1% of saponin (Sigma, Batch: BCBJ8417V), cells were incubated for2 h with mouse monoclonal antibody anti microtubule-associated-protein 2(MAP-2; Sigma, Batch: 063M4802) at dilution of 1/400 in PBS (Panbiotech, Batch: 4831114) containing 1% foetal calf serum (Invitrogen,Batch: 41Q7218K) and 0.1% of saponin. This antibody was revealed withAlexa Fluor 488 goat anti-mouse IgG (Molecular probe, Batch: 1572559) atthe dilution of 1/400 in PBS containing 1% foetal calf serum and 0.1% ofsaponin for 1 H at room temperature.

Analysis of Total Number of Neurons

The immunolabeled cultures were automatically examined with ImageXpress(Molecular device USA) at ×20 magnification. For each condition, 30automatically fields per well (representing ˜80% of the total surface ofthe well) from 3 wells were analyzed. The total number of neurons wasautomatically analyzed using Custom module editor (Molecular Devices,USA). Data were expressed in percentage of control conditions (nointoxication, no Amyloid-beta 1-42=100%) in order to express the A-beta1-42 injury. All values were expressed as mean+/−SEM (s.e. mean) (n=3wells per condition per culture).

1.10—In Vitro Model of Leukodystrophy (PMD): Overexpression of MutatedPLP1 and DM20 in Human Cell Line

One day before transfection, 293T cells were plated at 300,000 cells/mL.293T cells were transfected with PLP1 and DM20 mutant constructs usingLipofectamine 2000 according to manufacturer's procedure. Aftertransfection, cells were treated with molecules or left untreated. As acontrol, cells were transfected with native forms of the proteins. 48 hlater, cellular lysates were harvested. Protein accumulation wasassessed by western-blot.

1.11—In Vitro Model of Type 2 Diabetes: Min6 and INS1 Cell Lines

Cytoprotection from ER Stress

Cells were plated in 96 well plates at a density of 0.5·10⁶ cells/mL forMin6 cell line, 0.4·10⁶ cells/mL for INS1 cell line the day before thetreatment. ER stress was elicited by addition of 2.5 μg/mL tunicamycin(Sigma Aldrich) together with phosphatases inhibitors. Media werechanged 6 h later with fresh media and the cytoprotection was maintainedby the addition of phosphatases inhibitors. Cell viability was assessedby measuring the reduction of WST-8 into formazan using Cell CountingKit-8 (Sigma) according to the supplier's recommendation, 72 h aftertunicamycin treatment.

Protection Against Accumulation of Misfold Prone Insulin^(Akita)

Min6 cells were nucleofected with Insulin^(Akita) mutant constructs andseeded in 96 well-plates at 300,000 cells/mL and 24 h later, cells weretreated with molecules or left untreated. As a control, cells werenucleofected with non-relevant plasmid. 6 days later, a selective agentwas added (G418). Cell viability was assessed by measuring the reductionof WST-8 into formazan using Cell Counting Kit-8 (Sigma) according tothe supplier's recommendation, 9 days after treatment.

1.12—In Vitro Inflammation/Infection Disease Model: Poly I:C InducedMouse Embryonic Fibroblasts

Experimental Protocols

Mouse Embryonic Fibroblasts (MEFs) were lipofected with poly I:C andtreated with two concentrations of compounds of the invention (25 μM)for 6 h. After 6 h of culture, elF2alpha-phosphorylation (eIF2a-P) andPPP1R15A (GADD34) expression was monitored by western blotting, whiletype-I Interferon(IFN)-beta production was quantified in culturesupernatants by ELISA. Control (nt) and poly I:C/DMSO are respectivelynegative and positive controls. Poly I:C (polyinosinic:polycytidylicacid or polyinosinic-polycytidylic acid sodium salt) is animmunostimulant used to simulate viral infections. Poly I:C which isstructurally similar to double-stranded RNA, is known to interact withtoll-like receptor 3 which is expressed in the intracellularcompartments of B-cells and dendritic cells. Guanabenz (25 μM) was usedas reference inhibitory compound.

Cell Culture

MEFs were cultured in DMEM, 10% FCS (HyClone, Perbio), 100 units/mlpenicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1×MEM non-essentialamino acids and 50 μM 2-mercaptoethanol. MEFS were treated for theindicated time with 10 μg/ml poly I:C (InvivoGen) in combination withlipofectamine 2000 (Invitrogen).

Immunoblotting

Cells were lysed in 1% Triton X-100, 50 mM Hepes, 10 mM NaCl, 2.5 mMMgCl₂, 2 mM EDTA, 10% glycerol, supplemented with Complete Mini ProteaseInhibitor Cocktail Tablets (Roche). Protein quantification was performedusing the BCA Protein Assay (Pierce). 25-50 μg of Triton X-100-solublematerial was loaded on 2%-12% gradient or 8% SDS-PAGE beforeimmunoblotting and chemi-luminescence detection (SuperSignal West PicoChemi-luminescent Substrate, Pierce). Rabbit polyclonal antibodiesrecognizing GADD34 (C-19) were from Santa Cruz Biotechnology andanti-elF2alpha[pS⁵²] were from Invitrogen.

Elisa

IFN-beta quantification in culture supernatant was performed using theMouse Interferon Beta ELISA kit (PBL Interferon Source) according tomanufacturer instructions.

1.13—Hypoxia-Induced Apoptosis in Cultured Neonatal Rat Cardiomyocytes

Cell Culture

Primary cultures of neonatal rat cardiomyocytes were obtained from theventricles of 1-day-old Sprague Dawley rats (Janvier, France). The ratswere euthanized and their hearts excised. Hearts cut into small pieces(1-2 mm³) and enzymatically digested using the Neonatal HeartDissociation Kit rat and the gentleMACS™ Dissociator (MiltenyiBiotec,Germany). After dissociation, the homogenates were filtered (70 μm) toobtain a single-cell suspension. Isolated cells were collected bycentrifugation and resuspended in Dulbecco's Modified Eagle's Medium(DMEM) containing 10% horse serum (HS), 5% fetal bovine serum (FBS) and1% penicillin/streptomycin. Cultures were enriched with myocytes bypre-plating for 90 min to deplete the population of non-myocytes.Non-attached cells were plated onto 6- or 96-well plates at anappropriate cell density. The cells were cultured at 37° C. in 95%air/5% CO₂ for 24 h. Then the culture medium was exchanged with freshDMEM containing 1% FBS and different concentrations of test compoundthirty minutes before incubation in a normal or a hypoxic (N₂/CO₂,95%/5%; 0.3% O₂) culture chamber.

Treatment with Test Compound

Purified neonatal rat cardiomyocytes were seeded in a 96-well plate at10⁶ cells/2 mL for flow cytometry experiments.

After 24 hours, the cardiomyocytes were treated with differentconcentrations of test compound in culture medium with 0.1% DMSO. Thepositive controls cells were treated with culture medium (0.1% DMSO).Thirty minutes after starting the treatments, the cells were incubatedin the hypoxic culture chamber (N₂/CO₂, 95%/5%; final measured O₂: 0.3%)for 36 hours.

The negative controls cells were left in normoxic conditions at 37° C.with culture medium (1% FBS, 0.1% DMSO) for the same time periods.

Apoptotic Cell Measurement

At the end of the treatment period, flow cytometry were performed tomeasure the amount of apoptotic cells. The Annexin V-fluoresceinisothiocyanate (FITC) apoptosis detection kit from Miltenyi was used.Cells were washed twice with PBS and re-suspended in binding buffer.FITC-Annexin V and propidium iodide were added according to themanufacturer's protocol. The mixture was incubated for 15 min in thedark at room temperature, and cellular fluorescence was then measured byFACS scan flow cytometry.

2—Results

2.1—Cytoprotection & Compound Selectivity

The results of the different assays ran with selected compounds of theinvention are shown below in Table 1.

As example, FIG. 1 represents the cytoprotective effect of compound 12after the stress induced by an exposure of tunicamycin.

TABLE 1 Cytoprotection from Functional Compound ER stress comparedadrenergic alpha2 No to guanabenz receptor assay 1 + 2 + 3 + 4 ++ 5 + 6++ EC50 > 33.3 μM 7 + 8 + 9 + 10 ++ EC50 > 33.3 μM 11 +++ EC50 > 0.7 μM 12 +++ EC50 > 33.3 μM 13 ++ EC50 > 33.3 μM 14 ++ 15 ++ EC50 > 33.3 μM16 + EC50 > 33.3 μM 17 +++ EC50 > 33.3 μM 19 +++ 21 ++ 23 ++

2.2—Multiple Sclerosis

FIG. 2 shows dose dependent protection of interferon-gamma injured ratoligodendrocytes by compounds 11, 12 and 17 of the invention.

These data show that the compounds of this invention are promisingeffective treatment of Multiple Sclerosis.

2.3—Parkinson's Disease (PD)

FIG. 3 shows dose dependent protection of rotenone injured primarymesencephalic rat neurons by compounds 5, 11 and 12 of the invention.

These data show that the compounds of this invention are promisingeffective treatment of synucleopathies, and more specificallyParkinson's disease.

2.4—Alzheimer Disease (AD) & Amyloidosis

FIG. 4 shows dose dependent protection of amyloid-beta 1-42 injuredprimary cortical rat neurons by compound 12 of the invention.

These data show that the compounds of this invention are promisingeffective treatment of Amyloidosis and more specifically Alzheimerdisease.

2.5—Leukodystrophy: Pelizaeus-Merzbacher Disease (PMD),

T181P and L223P mutations in PLP1 and DM20 proteins have been describedto cause a severe phenotype of Pelizaeus-Merzbacher disease (Strautniekset al. 1992, Am. J. Hum. Genet. 51 (4): 871-878; Gow and Lazzarini, 1996Nat Genet. 13(4):422-8).

The Compound 12 and 17 of the invention (5 microM) is able to preventthe accumulation of T181P mutated DM20 protein expressed in Human 293Tcell (FIG. 5).

These data show that the compounds of this invention, specificallycompounds 12 and 17, are promising effective treatment of demyelinatingdisorders like leukodystrophies, more specifically PMD.

2.6—Type 2 Diabetes

FIG. 6 represents the results of over expression of pre-pro-insulinbearing Akita mutation in Min6 cells with compound 16 of the invention.

The compounds 12, 16 and 17 at different concentrations prevent Min6insulinoma cell death associated with accumulation of misfolded proteininduced by 6 hour exposure to tunicamycin (FIG. 7)

The compounds 11, 12, 16 and 17 at different concentrations prevent INS1insulinoma cell death associated with accumulation of misfolded proteininduced by 6 hour exposure to tunicamycin (FIG. 8).

These data show that the compounds of the invention are promisingeffective treatment of pre-diabetes and diabetes, preferably type 2pre-diabetes and type 2 diabetes.

2.7—Infection-Related or Non-Infectious Inflammatory Conditions

Normal response of MEFs to poly I:C is characterized by PPP1R15Aexpression, increase in elF2alpha-P (variable in time and related to thelevels PPP1R15A expression) mediated by PKR activation and type-I IFNproduction (range 500 to 700 μg/ml). Knock out PPP1R15A−/−MEFs areunable to produce this cytokine in response to poly I:C.

The potency of compounds of the invention to inhibit PPP1R15A wasevaluated by measuring the increase of elF2alpha phosphorylation, thedecrease of PPP1R15A expression due to its own pharmacologicalinhibition resulting in general protein synthesis inhibition and type-IIFN production.

The evaluated compounds of the invention were found efficient at 25 μMto increase elF2alpha phosphorylation, to decrease of PPP1R15Aexpression and to prevent type-I IFN production. As example, FIG. 9shows the ability of compounds 6, 10, 11, 12, 15, 16 and 17 (at 25microM) to prevent type-I IFN production by mouse embryonic fibroblastslipofected with poly I:C.

These data show that the compounds of this invention are promisingeffective treatment of infection-related or non-infectious inflammatoryconditions.

2.8—Cardiac Ischemia

Compound 10 of the invention protects cultured neonatal ratcardiomyocytes from hypoxia-induced apoptosis (FIG. 10). These data showthat the compounds of this invention are promising effective treatmentof ischemia, specifically cardiac ischemia. Various modifications andvariations of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in the relevant fields areintended to be covered by the present invention.

1. A compound of formula (I) or a pharmaceutically acceptable saltthereof:

wherein: Hal=F, Cl, Br, I X is either —CR1= or —N═, Y is either —CR2= or—N═, Z is either —CR3= or —N═, W is either —CR4= or —N═, R1 is selectedfrom H, Hal, alkyl and O-alkyl; R2 is selected from H, Hal, alkyl,O-alkyl and C(O)R6; R3 is selected from H, Hal, alkyl and O-alkyl; R4 isH, Cl, F, I or Br; R5 is alkyl, cycloalkyl, aralkyl, alkenyl,cycloalkenyl, heterocyclyl, aryl, C(O)-alkyl, and C(O)-aryl, each ofwhich is optionally substituted with one or more R7 groups; R6 isselected from OH, O-alkyl, O-aryl, aralkyl, NH2, NH-alkyl, N(alkyl)2,NH-aryl, CF3, alkyl and alkoxy; each R7 is independently selected fromhalogen, OH, CN, COO-alkyl, aralkyl, heterocyclyl, S-alkyl, SO-alkyl,S02-alkyl, S02-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl)2,CF3, alkyl and alkoxy.
 2. A compound according to claim 1 wherein Hal isCl.
 3. A compound according to claim 1 wherein X is —CR1= and R1 is H orF.
 4. A compound use according to claim 1 wherein Y is —CR2= and R2 is Hor F.
 5. A compound according to claim 1 wherein Z═—CR3= and R3 is H orF.
 6. A compound according to claim 1 wherein W═—CR4= and R4 is H, Cl orF.
 7. A compound according to claim 1 wherein R5 is chosen from, alkenylor alkyl, each of which is optionally substituted with one or more R7groups chosen from halogen, OH, heterocyclyl, S-alkyl, SO-alkyl,SO₂-alkyl, Oalkyl.
 8. A compound according to claim 1 which is selectedfrom the following:

or acceptable salt thereof.
 9. A process for preparing a compound offormula (I) or pharmaceutically acceptable salts thereof according toclaim 1 comprising the step of reacting a compound of formula (A):

wherein R5 is defined as in claim 1, with a compound of formula (B):

wherein X, Y, Z, W and Hal are defined according to claim
 1. 10. Theprocess according to claim 9 which comprises a further step ofpurification.
 11. A pharmaceutical composition comprising a compound offormula (II):

wherein: Hal is F, Cl, Br, or I; X is either —CR1= or —N═, Y is either—CR2= or —N═, Z is either —CR3= or —N═, W is either —CR4= or —N═, R1 isselected from H, Hal, alkyl and O-alkyl; R2 is selected from H, Hal,alkyl, O-alkyl and C(O)R6; R3 is selected from H, Hal, alkyl andO-alkyl; R4 is H, Cl, F, I or Br; R5 is alkyl, cycloalkyl, aralkyl,alkenyl, cycloalkenyl, heterocyclyl, aryl, C(O)-alkyl, and C(O)-aryl,each of which is optionally substituted with one or more R7 groups; R6is selected from OH, O-alkyl, O-aryl, aralkyl, NH₂, NH-alkyl, N(alkyl)₂,NH-aryl, CF₃, alkyl and alkoxy; each R7 is independently selected fromhalogen, OH, CN, COO-alkyl, aralkyl, heterocyclyl, SO-alkyl, SO₂-alkyl,SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyland alkoxy; with a suitable pharmaceutically acceptable diluent,excipient or carrier.
 12. The pharmaceutical composition according toclaim 11 wherein Hal is Cl.
 13. A method for treating a diseaseassociated with protein misfolding stress and in particular with anaccumulation of misfolded proteins, comprising administering to apatient in need thereof, a compound of formula (II) according to claim11.
 14. The method according to claim 13 wherein the disease isassociated with the PPP1R15A pathway.
 15. The method according to claim13 wherein the disease is selected in the group of tauopathies chosenfrom Alzheimer disease, progressive supranuclear palsy, corticobasaldegeneration, frontotemporal lobar degeneration or frontotemporaldementia (FTD) (Pick's disease); synucleinopathies chosen fromParkinson's disease, dementia with Lewy bodies, pure autonomic failure,and multiple system atrophy; polyglutamine and polyalanine diseaseschosen from Huntington disease, spinobulbar muscular atrophy (or Kennedydisease), dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxiatype 1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3 (orMachado-Joseph disease), Spinocerebellar ataxia type 6, Spinocerebellarataxia type 7 and Spinocerebellar ataxia type 17, oculo-pharyngealmuscular dystrophy; demyelinating disorders like leukodystrophies,Charcot-Marie-Tooth disease and multiple sclerosis, cystic fibrosis,chosen from systemic lupus erythematosus, pancreatitis and sepsis,seipinopathies, lysosomal storage disorders, amyloidosis diseases,inflammation, metabolic disorders and cardio-vascular disorders chosenfrom adiposity, hyper-lipidemia, familial hyper-cholesterolemia,obesity, atherosclerosis, hypertension, heart diseases, cardiacischaemia, stroke, myocardial infraction, trans-aortic constriction,vascular stroke; osteoporosis, nervous system trauma, ischemia,osteoporosis, retinal diseases chosen from retinitis pigmentosa, retinalciliopathies, glaucoma, macular degeneration and aging.
 16. Thepharmaceutical composition according to claim 11 wherein X is —CR1= andR1 is H or F.
 17. The pharmaceutical composition according to claim 11wherein Y is —CR2= and R2 is H or F.
 18. The pharmaceutical compositionaccording to claim 11 wherein Z═—CR3= and R3 is H or F.
 19. Thepharmaceutical composition according to claim 11 wherein W═—CR4= and R4is H, Cl or F.
 20. The pharmaceutical composition according to claim 11wherein R5 is chosen from, alkenyl or alkyl, each of which is optionallysubstituted with one or more R7 groups chosen from halogen, OH,heterocyclyl, S-alkyl, SO-alkyl, SO₂-alkyl, Oalkyl.
 21. Thepharmaceutical composition according to claim 11 wherein the compound offormula (I) is selected from the following: